Value-added traits in grain and seed transformed with thioredoxin

ABSTRACT

Compositions and methods of use are provided herein to make and use transgenic plants with value-added traits.

This application claims the benefit of the filing date of applicationSer. No. 60/126,736, filed Mar. 29, 1999, pending; application Ser. No.60/127,198, filed Mar. 31, 1999 pending, application Ser. No.60/169,162, filed Dec. 6, 1999, pending; application Ser. No. 60/177,740filed Jan. 21, 2000, pending; and application Ser. No. 60/177,739, filedJan. 21, 2000, pending, all of which are expressly incorporated byreference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant 9803835 fromthe U.S. Department of Agriculture. The Government has certain rights tothis invention.

BACKGROUND OF THE INVENTION

Thioredoxins are small (about 12 kDa) thermostable proteins withcatalytically active disulfide groups. This class of proteins has beenfound in virtually all organisms, and has been implicated in myriadbiochemical pathways (Buchanan et al., 1994). The active site ofthioredoxin has two redox-active cysteine residues in a highly conservedamino acid sequence; when oxidized, these cysteines form a disulfidebridge (—S—S—) that can be reduced to the sulfhydryl (—SH) level througha variety of specific reactions. In physiological systems, thisreduction may be accomplished by reduced ferredoxin, NADPH, or otherassociated thioredoxin-reducing agents. The reduced form of thioredoxinis an excellent catalyst for the reduction of even the most intractabledisulfide bonds. Generally only one kind of thioredoxin is found inbacterial or animal cells. In contrast, photosynthetic organisms havethree distinct types of thioredoxin. Chloroplasts contain aferredoxin/thioredoxin system comprised of ferredoxin,ferredoxin-thioredoxin reductase and thioredoxins f and m, whichfunction in the light regulation of photosynthetic enzymes (Buchanan,1991; Scheibe, 1991). The other thioredoxin enzyme system is analogousto that established for animals and most microorganisms, in whichthioredoxin (h-type in plants) is reduced by NADPH and NADPH-thioredoxinreductase (NTR) (Johnson et al., 1987a; Florencio et al., 1988; Suske etal., 1979). The reduction of thioredoxin h by this system can beillustrated by the following equation:

Thioredoxin is a component of two types of enzyme systems in plants.Chloroplasts contain a ferredoxin/thioredoxin system comprised offerredoxin, ferredoxin-thioredoxin reductase and thioredoxins f and m,that are involved in the light regulation of photosynthetic enzymes(Buchanan, 1991; Scheibe, 1991). The other enzyme system, theNADP-thioredoxin system or NTS, is analogous to the system establishedfor animals and most microorganisms, in which thioredoxin (h-type inplants) is reduced by NADPH and NADPH-thioredoxin reductase (NTR)(Johnson et al., 1987a; Florencio et al., 1988; Suske et al., 1979).Thioredoxin h is widely distributed in plant tissues and exists inmitochondria, endoplasmic reticulum (ER) and cytosol (Bodenstein-Lang etal., 1989; Marcus et al., 1991).

Plant thioredoxin h is involved in a wide variety of biologicalfunctions. The presence of multiple forms of thioredexoin h protein hasalso been reported in plant seeds (Bestermann et al., 1983). In wheat,three different thioredoxin have been characterized (Vogt and Follman,1986). Thioredoxin h functions in the reduction of intramoleculardisulfide bridges of a variety of low molecular-weight, cystine-richproteins, including thionins (Johnson et al., 1987b), proteaseinhibitors and chloroform/methanol-soluble proteins (CM proteins oralpha-amylase inhibitors) (Kobrehel et al., 1991). It is likely thatcytoplasmic thioredoxins participate in developmental processes: forexample thioredoxin h has been shown to function as a signal to enhancemetabolic processes during germination and seedling development(Kobrehel et al., 1992; Lozano et al., 1996; Besse et al., 1996).Thioredoxin h has also been demonstrated to be involved inself-incompatibility in Phalaris coerulescens (Li et al., 1995) andBrassica napus (Bower et al., 1996). Several functions have beenhypothesized for rice thioredoxin h, which is believed to be involved intranslocation in sieve tubes (Ishiwatari et al., 1995).

The NTS has been shown to improve dough quality. The improvement indough strength and bread quality properties of poor-quality wheat flourresulting from the addition of thioredoxin (Wong et al., 1993; Kobrehelet al., 1994) may be attributable to the thioredoxin-catalyzed reductionof intramolecular disulfide bonds in the flour proteins, specificallythe glutenins, resulting in the formation of new intermoleculardisulfide bonds (Besse and Buchanan, 1997). Thus, the addition ofexogenous thioredoxin promotes the formation of a protein network thatproduces flour with enhanced baking quality. Kobrehel et al., (1994)have observed that the addition of thioredoxin h to flour ofnon-glutenous cereals such as rice, maize and sorghum promotes theformation of a dough-like product. Hence, the addition of exogenousthioredoxin may be used to produce baking dough from non-glutenouscereals.

In addition, it has been shown that reduction of disulfide proteinallergens in wheat and milk by thioredoxin decreases their allergenicity(Buchanan et al., 1997; del Val et al., 1999). Thioredoxin treatmentalso increases the digestibility of the major allergen of milk(β-lactoglobulin) (del Val et al., 1999), as well as other disulfideproteins (Lozano et al., 1994; Jiao et al., 1992). Therefore, themanipulation of the NTS offers considerable promise for production ofnutraceutical and pharmaceutical products. A more detailed discussion ofthe benefits of adding exogenous thioredoxin to food products ispresented in U.S. Pat. No. 5,792,506 to Buchanan et al.

cDNA clones encoding thioredoxin h have been isolated from a number ofplant species, including Arabidopsis thaliana (Rivera-Madrid et al.,1993; Rivera-Madrid et al., 1995), Nicotiana tabacum (Marty and Meyer,1991; Brugidou et al., 1993), Oryza sativa (Ishiwatari et al., 1995),Brassica napus (Bower et al., 1996), Glycine max (Shi and Bhattacharyya,1996), and Triticum aestivum (Gautier et al., 1998). More recently, twocDNA clones encoding wheat thioredoxin h have been isolated andcharacterized (Gautier et al., 1998). The Esherichia coli NTR gene hasbeen first isolated (Russel and Model, 1988) and the three-dimensionalstructure of the protein has been analyzed (Kuriyan et al., 1991). Someother NTR genes have been isolated and sequenced from bacteria, fungiand mammals. Recently, Jacquot et al., (1994) have reported a successfulisolation and sequencing of two cDNAs encoding the plant A. thalianaNTRs. The subsequent expression of the recombinant A. thaliana NTRprotein in E. coli cells (Jacquot et al., 1994) and its first eukaryoticstructure (Dai et al., 1996) have also been reported.

Here we disclose value-added traits in transgenic grains, such as barley(Cho et al., 1999b)., wheat, and sorghum, overexpressing thioredoxin

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the thioredoxin h constructs used for transformation.

FIG. 2 shows the thioredoxin activity profile of various barley grainstransformed with wheat thioredoxin gene (wtrxh).

FIG. 3 shows the effects of heat treatment on thioredoxin activity ofcrude extracts from barley grains.

FIG. 4A-B shows a western blot analysis of extract from segregating T₁barley grain of stable transformants containing wtrxh. Panel A: lanes 1and 6, control barley extract (cv. Golden Promise); lane 2, bread wheatextract (Triticum aestivum, cv. Capitole); lane 3, extract from GPdBhssBarWtrx 22; lane 4, extract from GPdBhssBarWtrx 29; lane 5, extract fromGPdBhBarWtrx 2. Panel B: lane 1, GPdBhBaarWtrx 2; lane 2 control barleyextract. W, wheat; B, barley.

FIG. 5 shows western blot analysis of extracts of T₁ T₂ and T₃ barleygrain transformed with wtrxh. Forty micrograms of soluble proteinsextracted from 10-20 grains of each line were fractionated by SDS/PAGE.Lane 1, wheat germ thioredoxin h; lane 2, nontransgenic control ofGP4-96; lane 3, null segregant T₂ grain of GPdBhssBarWtrx-29-11-10; lane4, heterozygous T₁ grain of GPdBhssBarWtrx-29; lane 5, homozygous T₂grain of GPdBhssBarWtrx-29-3; lane 6, homozygous T₂ grain ofGPdBhssBarWtrx-29-3-2; lane 7, prestained standards (aprotinin, 0.9 kDa;lysozyme, 17.8 kDa; soybean trypsin inhibitor, 30.6 kDa; carbonicanhydrase, 41.8 kDa; BSA, 71 kDa).

FIG. 6 shows the nucleic acid sequence of the B1-hordein promoter andthe 57 base pair B1-hordein signal sequence (underlined).

FIG. 7 shows the nucleic acid sequence of the D-hordein promoter and the63 base pair D-hordein signal sequence (underlined).

FIG. 8A-C shows the effect of overexpressed thioredoxin h on pullulanaseactivity in transgenic barley grain during germination and seedlingdevelopment. A homozygous line, GPdBhssBarWtrx-29-3, and a nullsegregant, GPdBhssBarWtrx-29-11-10, were used for the pullulanaseassays. Panel A: Pullulanase was assayed spectrophotometrically bymeasuring the dye released from red pullulan substrate at 534 nm. PanelB: Pullulanase was separated on native 7.5% polyacrylamide gelscontaining the red pullulan substrate. Activity, identified bycomparison with purified barley pullulanase, is seen as clear areas thatdeveloped on incubating the gel in 0.2 M succinate buffer, pH 6.0, for 1hr at 37° C. Panel C: The gel in Panel B was scanned and analyzed byintegration of the activity bands.

FIG. 9A-D shows the change in the activity and abundance of amylases intransgenic and null segregant barley grains during germination andseedling development based on an activity gel. Panel A: abundance ofalpha-amylases in null segregant based on western blot. Panel B: Totalamylase activity in null segregant. Panel C: abundance of alpha-amylasesin thioredoxin overexpressing grains. Panel D: total amylase activity inthioredoxin overexpressed grains.

FIG. 10 shows the effect of overexpressed thioredoxin h on the activityof the major form of alpha-amylase during germination and seedingdevelopment. The size of the major alpha-amylase activity band in FIG. 9was estimated by its rate of mobility during electrophoresis.

FIG. 11A-B shows the effect of overexpressed thioredoxin h on theabundance of alpha-amylase A and B isozymes during germination andseedling development. The figure represents western blots of IEF gelsdeveloped for the null segregant and transgenic barley grains. Panel A:Null segregant. Panel B: Transgenic with thioredoxin overexpressed.

FIG. 12 depicts the DNA constructs used for wheat transformation.

FIG. 13 shows the endosperm-specific expression of barley D-hordeinpromoter sgfp(S65T) in transgenic wheat plants. Transgenic endosperm isat the right, transgenic embryo is at the left.

FIG. 14 shows the PCR analysis of genomic DNA from transgenic wheatplants.

FIG. 15A-B shows wheat thioredoxin h-overexpressing wheat lines screenedby western blot analyses. Panel A: T₀ wheat lines. Panel B T₃ homozygousline.

FIG. 16 shows the effect of thioredoxin reduction on digestion of wheatglutenins by trypsin.

FIG. 17 shows the effect of thioredoxin reduction on digestion of wheatglutenins by pancreatin.

FIG. 18 show the effect of NTR on the reduction of proteins in extractsof transgenic wheat overexpressing thioredoxin h verses a nullsegregant.

FIG. 19 shows the effect of overexpressed thioredoxin h on allergenicityof proteins from wheat grain.

FIG. 20 shows the barley thioredoxin h nucleotide and amino acidsequence (SEQ ID NO:24, SEQ ID NO:26, respectively).

FIG. 21 shows the effect of overexpressed wheat thioredoxin h on thegermination of null segregant and transgenic (homozygous) barley grains.

FIG. 22 shows the relative redox status of protein fractions intransgenic barley grain overexpressing wheat thioredoxin h in comparisonto the null segregant in dry and germination grain.

FIG. 23 shows the effect of glucose-6-phosphate dehydrogenase on thereduction of proteins in extracts of transgenic wheat grainoverexpressing thioredoxin h in the presence of glucose 6-phosphate andArabidopsis NTR:+/−NTR.

FIG. 24 shows the effect of glucose-6-phosphate dehydrogenase on thereduction of proteins in extracts of extracts of null segregant derivedfrom wheat grain overexpressing thioredoxin h in the presence of glucose6-phosphate and Arabidopsis NTR:+/+NTR.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but it is understood that thecomplementary strand is included by any reference to the displayedstrand. SEQ ID NO:1 shows the nucleic acid sequence of the barleyB1-hordein promoter and signal sequence. SEQ ID NO:2 shows the aminoacid sequence of the barley B1-hordein signal sequence. SEQ ID NO:3shows the nucleic acid sequence of the barley D-hordein promoter andsignal sequence. SEQ ID NO:4 shows the ammo acid sequence of the barleyD-hordein signal sequence. Other sequences are identified below.

SUMMARY OF THE INVENTION

The present invention provides recombinant nucleic acids encodingthioredoxin and methods of use to produce transgenic plantsoverexpressing thioredoxin. Indeed, given the powerful reducing activityof thioredoxin, over-expression of this protein in a plant cell would beanticipated to have a serious . detrimental effect on the cell. However,the inventors have discovered that thioredoxin can be expressed at ahigh level in plants, particularly cereal grains, without affecting theviability of the cells in which the protein is expressed, or the seedsthemselves. By way of example, in certain embodiments the inventors haveintroduced a wheat thioredoxin gene (wtrxh) into wheat. Seeds of thetransgenic-wheat plants can show an increase thioredoxin specificactivity in comparison to non-transgenic-wheat plants.

The invention thus provides transgenic plants, wherein at least a partof a plant has an elevated level of thioredoxin protein and/orthioredoxin specific activity compared to the homologous part ofnon-transgenic plants of the same species. The level of thioredoxinspecific activity in the parts of the transgenic plants may be at leastabout two times greater than the parts of non-transgenic plants of thatspecies. While the invention is applicable to any plant species, it willbe particularly beneficial as applied to the monocotyledons, for examplecereal crops including, but not limited to rice, barley, wheat, oat,maize, rye, sorghum, millet, and triticale and the dicotyledonsincluding, but not limited to soybeans, lima beans, tomato, potato,soybean, cotton, tobacco. In a preferred embodiment, thioredoxinspecific activity is increased in the seeds of the transgenic plant.

Thioredoxin over-expression in a desired part of a plant, for example, aseed, is achieved by use of a seed-specific promoter operably linked tothe thioredoxin coding sequence. In this example, “seed-specific”indicates that the promoter has enhanced activity in seeds compared toother plant tissues; it does not require that the promoter is solelyactive in the seeds. However, given the nature of the thioredoxinprotein, it may be advantageous to select a seed-specific promoter thatin some cases causes little or no protein expression in tissues otherthan seeds. In certain embodiments, the seed-specific promoter that isselected is a seed maturation-specific promoter. The use of promotersthat confer enhanced expression during seed maturation (such as thebarley hordein promoters) may result in even higher levels ofthioredoxin expression in the maturing seed.

In an alternative embodiment, thioredoxin is overexpressed in the root,stem, tuber, fruit, leaf, flower, pollen etc or any one or more parts ofa plant at the discretion of the practitioner.

In one embodiment of the invention, the provided transgenic plantscomprise a recombinant nucleic acid molecule having a structure: P-T,wherein P is a seed-specific promoter, and T is an nucleic acid moleculeencoding a thioredoxin polypeptide. In particular embodiments, theseed-specific promoter is a barley hordein gene promoter, such as abarley B1-hordein promoter, a barley D-hordein promoter or a maizeembryo specific globulin promoter.

In another embodiment of the invention, the transgenic plants comprise arecombinant nucleic acid molecule having a structure: P-SS-T, wherein Pis a seed-specific promoter, T is an nucleic acid molecule encoding athioredoxin polypeptide and SS is a nucleic acid molecule that encodes asignal peptide that targets expression of the thioredoxin polypeptide toan intracellular body, and wherein P, SS and T are operably linked.Evidence presented herein indicates that the presence of the signalpeptide can further enhance the level of thioredoxin expression in thetransgenic plants. Suitable signal peptides include, but are not limitedto, barley B1- and D-hordein signal peptides.

Parts of the transgenic plants overexpressing thioredoxin as provided bythe invention may be harvested for direct processing into food products.For example, the seeds may be ground using conventional means to produceflour. Alternatively, the seeds or other plant parts may be used as asource of thioredoxin, which can be extracted from the immature ormature transgenic plant by standard protein extraction methods.Alternatively, crudely processed seed material may be used directly as asource of thioredoxin. Thus, another aspect of the invention is a methodof producing thioredoxin protein, the method comprising harvestingthioredoxin from the seed of a transgenic plant having an elevated levelof thioredoxin in its seeds.

Accordingly, in another aspect the invention provides an improved edibleproducts for human and animal consumption, for example increaseddigestibility and/or reduced allergenicity and dough having increasedstrength and volume in comparison to dough produced from non-transgenicplant of the same species.

In yet another aspect, the invention provides of methods of making atransgenic plant having reduced allergenicity, increased digestibility,increased redox state (increased SH:SS ratio), in comparison to anon-transgenic plant of the same species.

In still yet another aspect, the invention provide a transgenic plantcomprising a nucleic acid encoding A. thaliana NTR.

These and other aspects of the invention are further illustrated by thefollowing description and Examples.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes V published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology. a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Ausubel et al. (1987)Current Protocols in Molecular Biology, Green Publishing; Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.

In order to facilitate review of the various embodiments of theinvention, the following definitions are provided:

Thioredoxin protein or Thioredoxin polypeptide: A large number of plant,animal, and microbial thioredoxin proteins or polypeptides have beencharacterized, and the genes encoding many of these proteins have beencloned and sequenced. The present invention is preferably directed tothe use of thioredoxin h proteins, although other thioredoxin proteinsmay also be employed to produce transgenic plants as described herein.Among the thioredoxin h proteins from plants that have been described todate are thioredoxin h proteins from Arabidopsis thaliana (Rivera-Madridet al., 1993; Rivera-Madrid et al., 1995), Nicotiana tabacum (Marty andMeyer, 1991; Brugidou et al., 1993), Oryza sativa (ishiwatari et al.,1995), Brassica napus (Bower et al., 1996), Glycine max (Shi andBhattacharyya, 1996), and Triticum aestivum (Gautier et al., 1998). Theamino acid sequences of these and other thioredoxin h proteins, and thenucleotide sequence of cDNAs and/or genes that encode these proteins,are available in the scientific literature and publicly accessiblesequence databases. For example, a cDNA encoding thioredoxin h fromPicea mariana is described in accession number AF051206 (NID g2982246)of GenBank, and located by a search using the Entrez browser/nucleotidesequence search of the National Center for Biotechnology Informationwebsite, www.ncbi.nlm.nih.gov. The cDNA encoding the Triticum aestivumthioredoxin h protein used in the Examples described below is describedon the same database under accession number X69915 (NID g2995377).

The present invention may be practiced using nucleic acid sequences thatencode full length thioredoxin h proteins, as well as thioredoxin hderived proteins that retain thioredoxin h activity. Thioredoxin hderived proteins which retain thioredoxin biological activity includefragments of thioredoxin h, generated either by chemical (e.g.enzymatic) digestion or genetic engineering means; chemicallyfunctionalized protein molecules obtained starting with the exemplifiedprotein or nucleic acid sequences, and protein sequence variants, forexample allelic variants and mutational variants, such as those producedby in vitro mutagenesis techniques, such as gene shuffling (Stemmer etal., 1994a, 1994b). Thus, the term “thioredoxin h protein” encompassesfull length thioredoxin h proteins, as well as such thioredoxin hderived proteins that retain thioredoxin h activity.

Thioredoxin protein may be quantified in biological samples (such asseeds) either in terms of protein level, or in terms of thioredoxinactivity. Thioredoxin protein level may be determined using a westernblot analysis followed by quantitative scanning of the image asdescribed in detail below. Thioredoxin activity may be quantified usinga number of different methods known in the art. Preferred methods ofmeasuring thioredoxin biological activity attributable to thioredoxin hin plant extracts include NADP/malate dehydrogenase activation (Johnsonet al., 1987a,b) and reduction of 2′,5′-dithiobis(2-nitrobenzoic acid)(DTNB) via NADP-thioredoxin reductase (Florencio et al., 1988; U.S. Pat.No. 5,792,506). Due to the potential for interference fromnon-thioredoxin h enzymes that use NADPH, accurate determination ofthioredoxin h activity should preferably be made using partiallypurified plant extracts. Standard protein purification methods (e.g.(NH₄)₂SO₄ extraction or heat) can be used to accomplish this partialpurification. The activity of thioredoxin h may also be expressed interms of specific activity, i.e., thioredoxin activity per unit ofprotein present, as described in more detail below.

In another embodiment, thioredoxin may be expressed in terms ofthioredoxin content, such as, mass/mass tissue (i.e., μg/gram tissue) ormass/mass soluble protein (i.e., μg/mg soluble protein)

Promoter: A regulatory nucleic acid sequence, typically located upstream(5′) of a gene that, in conjunction with various cellular proteins, isresponsible for regulating the expression of the gene. Promoters mayregulate gene expression in a number of ways. For example, theexpression may be tissue-specific, meaning that the gene is expressed atenhanced levels in certain tissues, or developmentally regulated, suchthat the gene is expressed at enhanced levels at certain times duringdevelopment, or both.

In a preferred embodiment, a transgene of the invention is expressed inan edible part of a plant. By “edible” herein is meant at least a partof a plant that is suitable for consumption by humans or animals (fish,crustaceans, isopods, decapods, monkeys, cows, goats, pigs, rabbits,horses, birds (chickens, parrots etc). Accordingly, “edible” embracesfood for human consumption and feed for animal consumption and includes,for example, dough, bread, cookies, pasta, pastry, beverages, beer, foodadditives, thickeners, malt, extracts made from an edible part ofplants, animals feeds, and the like. An edible part of a plant includesfor example, a root, a tuber, a seed, grain, a flower, fruit, leaf etc.The skilled artisan is aware that expression of the transgene iseffected in any tissue, organ or part of a plant by employing a promoterthat is active in the selected part of the plant the transgene is to beexpressed. In a preferred embodiment the transgene is expressed in aseed, preferably under control of a seed- or grain-specific promoter.

The expression of a transgene in seeds or grains according to thepresent invention is preferably accomplished by operably linking aseed-specific or grain-specific promoter to the nucleic acid moleculeencoding the transgene protein. In this context, “seed-specific”indicates that the promoter has enhanced activity in seeds compared toother plant tissues; it does not require that the promoter is solelyactive in the seeds. Accordingly, “grain-specific” indicates that thepromoter has enhanced activity in grains compared to other planttissues; it does not require that the promoter is solely active in thegrain. Preferably, the seed- or grain-specific promoter selected will,at the time when the promoter is most active in seeds, produceexpression of a protein in the seed of a plant that is at least abouttwo-fold greater than expression of the protein produced by that samepromoter in the leaves or roots of the plant. However, given the natureof the thioredoxin protein, it may be advantageous to select a seed- orgrain-specific promoter that causes little or no protein expression intissues other than seed or grain. In a preferred embodiment, a promoteris specific for seed and grain expression, such that, expression in theseed and grain is enhanced as compared to other plant tissues but doesnot require that the promoter be solely activity in the grain and seed.In a preferred embodiment, the promoter is “specific” for a structure orelement of a seed or grain, such as an embryo-specific promoter. Inaccordance with the definitions provided above, an embryo-specificpromoter has enhanced activity in an embryo as compared to other partsof a seed or grain or a plant and does not require its activity to belimited to an embryo. In a preferred embodiment, the promoter is“maturation-specific” and accordingly has enhanced activitydevelopmentally during the maturation of a part of a plant as comparedto other parts of a plant and does not require its activity to belimited to the development of a part of a plant.

A seed- or grain-specific promoter may produce expression in varioustissues of the seed, including the endosperm, embryo, and aleurone orgrain. Any seed- or grain-specific promoter may be used for thispurpose, although it will be advantageous to select a seed- orgrain-specific promoter that produces high level expression of theprotein in the plant seed or grain. Known seed- or grain-specificpromoters include those associated with genes that encode plant seedstorage proteins such as genes encoding: barley hordeins, riceglutelins, oryzins, or prolamines; wheat gliadins or glutenins; maizezeins or glutelins; maize embryo-specific promoter; oat glutelins;sorghum kafirins; millet pennisetins; or rye secalins.

The barley hordein promoters (described in more detail below) are seed-or grain-specific promoters that were used in the illustrative Examples.

In certain embodiments, the seed- or grain-specific promoter that isselected is a maturation-specific promoter. The use of promoters thatconfer enhanced expression during seed or grain maturation (such as thebarley hordein promoters) may result in even higher levels ofthioredoxin expression in the seed.

By “seed or grain-maturation” herein refers to the period starting withfertilization in which metabolizable food reserves (e.g., proteins,lipids, starch, etc.) are deposited in the developing seed, particularlyin storage organs of the seed, including the endosperm, testa, aleuronelayer, embryo, and scutellar epithelium, resulting in enlargement andfilling of the seed and ending with seed desiccation.

Members of the grass family, which include the cereal grains, producedry, one-seeded fruits. This type of fruit, is strictly speaking, acaryopsis but is commonly called a kernel or grain. The caryopsis of afruit coat or pericarp, which surrounds the seed and adhere tightly to aseed coat. The seed consists of an embryo or germ and an endospermenclosed by a nucellar epidermis and a seed coat. Accordingly the graincomprises the seed and its coat or pericarp. The seed comprises theembryo and the endosperm. (R. Carl Hoseney in “Principles of CerealScience and Technology”, expressly incorporated by reference in itsentirety).

Hordein promoter: A barley promoter that directs transcription of ahordein gene in barley seeds or grains A number of barley hordein genesand associated promoters have been described and characterized,including those for the B-, C-, D-, and Gamma-hordeins (Brandt et al.,1985; Forde et al., 1985; Rasmussen and Brandt, 1986, Sørensen et al.,1996). The activities of these promoters in transient expression assayshave also been characterized (Entwistle et al., 1991; Muller andKnudesen, 1993; Sørensen et al, 1996). While any hordein promoter may beemployed for this invention, the specific Examples provided describe theuse of the promoter sequences from the B₁- and D-hordein genes ofbarley. The nucleic acid sequences of the barley B₁- and D-hordein genesare shown in SEQ ID NOs:1 and 3, respectively and in FIGS. 6 and 7 (thepromoter region excludes those nucleotides that encode the hordeinsignal peptide that is shown underlined). Sørensen et al., (1996)describes plasmids that comprise the B₁- and D-hordein promotersoperably linked to a beta-glucuronidase gene (uidA; gus) and theAgrobacterium tumefaciens nopaline synthase 3′ polyadenylation site(nos). These plasmids may be conveniently utilized as sources of boththe hordein promoters and the nos polyadenylation site.

One of skill in the art will appreciate that the length of the hordeinpromoter region may also be greater or less than the sequences depictedin FIGS. 6 and 7. For example, additional 5′ sequence from the hordeingene upstream region may be added to the promoter sequence, or bases maybe removed from the depicted sequences. However, any hordein promotersequence must be able to direct transcription of an operably linkedsequence in plant seed or grain. The ability of a barley hordeinpromoter to direct transcription of a protein in a plant seed mayreadily be assessed by operably linking the promoter sequence to an openreading frame (ORF) that encodes a readily detectable protein, such asthe gus ORF, introducing the resulting construct into plants and thenassessing expression of the protein in seeds of the plant (see Sørensenet al., 1996). A hordein promoter will typically confer seed-specificexpression, meaning that expression of the protein encoded by theoperably linked ORF will generally be at least about twice as high(assessed on an activity basis) in seeds of the stably transfected plantcompared to other tissues such as leaves. More usually, the hordeinpromoter will produce expression in seeds that is at least about 5 timeshigher than expression in other tissues of the plant.

Functional homologs of the barley hordein promoters disclosed herein maybe obtained from other plant species, such as from other monocots,including wheat, rice and corn. Such homologs may have specified levelsof sequence identity with the prototype hordein promoters (e.g., atleast 40% sequence identity). The functional homologs retain hordeinpromoter function, i.e., retain the ability to confer seed- orgrain-specific expression on operably linked ORFs when introduced intoplants (Marris et al., 1988; Mena et al., 1998). Accordingly, wherereference is made herein to a hordein promoter, it will be understoodthat such reference includes not only nucleic acid molecules having thesequences of the prototypical sequences disclosed herein (or variationson these sequences), but also promoters from hordein gene homologs. Alsoincluded within the scope of such terms are molecules that differ fromthe disclosed prototypical molecules by minor variations. Such variantsequences may be produced by manipulating the nucleotide sequence ofhordein promoter using standard procedures such as site-directedmutagenesis or the polymerase chain reaction. Preferably, the seed- orgrain-specificity of the promoter is retained. Examples of dicotpromoters that can be used include for example soybean glycinins andcon-glycinins, and kidney bean phaseolin promoters.

Signal peptide: As described in the Examples below, the inventors havediscovered that the level of expression of thioredoxin in seed or graincan be enhanced by the presence of a signal peptide. In one of theExamples described below, the B1 hordein signal peptide was utilized. Inparticular, it was discovered that the expression of thioredoxin proteinin seed or grain is enhanced when the ORF encoding the protein isoperably linked to both a hordein promoter and a hordein signal sequenceencoding the signal peptide. (For convenience, the nucleic acid sequenceencoding a signal peptide is referred to herein as a signal sequence.)While not wishing to be bound by theory, it is proposed that the hordeinsignal peptide directs expression of the thioredoxin protein to aprotected subcellular location, such as a vacuole or protein body. It isfurther proposed that proteins directed to such vacuoles are protectedfrom proteolysis during certain stages of seed or grain maturation. Inaddition, the sequestration of the thioredoxin protein to such alocation may also serve to protect the maturing seeds or grain fromdetrimental effects associated with thioredoxin over-expression.

The hordein signal peptide typically comprises about the first 15-25amino acids of the hordein gene ORF, more usually about 18-21 aminoacids. The nucleotide and amino acid sequences of the hordein signalsequence and peptide of the prototypical barley B1- and D-hordein genesare shown in SEQ ID NOS: 1-4 and FIGS. 6 and 7. One of skill in the artwill appreciate that while the B1-hordein signal sequence and signalpeptide are utilized in the examples described below, the invention isnot limited to these specific sequences. For example, homologoussequences may be used as effectively, as may sequences that differ inexact nucleotide or amino acid sequences, provided that such sequencesresult in enhanced levels of the encoded protein in immature seed orgrain. Typically, “enhanced expression” will be expression that is abouttwice that observed with an equivalent construct lacking the signalsequence. Accordingly, the term “hordein signal sequence” and “hordeinsignal peptide” includes not only the particular sequences shown herein,but also homologs and variants of these sequences.

Furthermore, the invention is not limited to the use of hordein signalpeptides. Other signal peptides that serve to localize the thioredoxinco-translationally or post-translationally to a selected seed, grain orcell compartment may be employed. Other such signal sequences includethose associated with storage proteins in maize, rice, wheat, soybeans,beans, and tobacco (see for example: Bagga et al., 1997; Torrent et al.,1997; Wu et al., 1998; Zheng et al., 1995; Grimwade et al., 1996; Conradet al., 1998; and Takaiwa et al., 1995.)

Starch: A polysaccharide made up of a chain of glucose units joined byalpha-1,4 linkages, either unbranched (amylose) or branched(amylopectin) at alpha-1,6-linkages.

Dextran: Any of a variety of storage polysaccharides, usually branched,made of glucose residues joined by alpha-1,6 linkages.

Dextrin or Limit Dextrin: Any of a group of small solublepolysaccharides, partial hydrolysis products of starch, usually enrichedin alpha-1,6-linkages.

Germination: A resumption of growth of a plant embryo in favorableconditions after seed maturation and drying (dessication), and emergenceof young shoot and root from the seed.

Allergen: An antigenic substance that induces an allergic reaction in asusceptible host. Accordingly, a susceptible host has an immune status(hypersensitivity) that results in an abnormal or harmful immunereaction upon exposure to an allergen. In a preferred embodiment, thetransgenic grains of the invention have reduced allergenicity incomparison to nontransgenic grains. The immune reaction can be immediateor delayed; cell mediated or antibody mediated; or a combinationthereof. In a preferred embodiment, the allergic reaction is animmediate type hypersensitivity.

Digestion: By “digestion” herein is meant the conversion of a moleculeor compound to one or more of its components. Accordingly,“digestibility” relates to the rate and efficiency at which theconversion to one or more of its components occurs. In a preferredembodiment a “digestible compound” is, for example, a food, that isconverted to its chemical components by chemical or enzymatic means. Forexample, dextran is converted to dextrin, polysaccharide,monosaccharides, limit dextrin etc; a protein is converted to apolypeptides, oligopeptides, amino acids, ammonia etc.; a nucleic acidis converted to oligonucleotides, nucleotides, nucleosides, purine,pyrimidines, phosphates etc. In a preferred embodiment, the transgenicgrains of the invention have increased digestibility, i.e. are moreefficiently or rapidly digested in comparison to nontransgenic grain.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences is expressed in terms of sequence identity (or,for proteins, also in terms of sequence similarity). Sequence identityis frequently measured in terms of percentage identity; the higher thepercentage, the more similar the two sequences are. As described above,homologs and variants of the thioredoxin nucleic acid molecules, hordeinpromoters and hordein signal peptides may be used in the presentinvention. Homologs and variants of these nucleic acid molecules willpossess a relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (1981); Needleman and Wunsch (1970); Pearson and Lipman(1988); Higgins and Sharp (1988); Higgins and Sharp (1989); Corpet etal., (1988); Huang et al., (1992); and Pearson et al., (1994). Altschulet al., (1994) presents a detailed consideration of sequence alignmentmethods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. It can be accessed athttp://www.ncbi.nlm.nih.gov/BLAST. A description of how to determinesequence identity using this program is available athttp://www.nchi.nlm.nih.gov/BLAST/blast.help.html.

Homologs of the disclosed protein sequences are typically characterizedby possession of at least 40% sequence identity counted over the fulllength alignment with the amino acid sequence of the disclosed sequenceusing the NCBI Blast 2.0, gapped blastp set to default parameters. Theadjustable parameters are preferably set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 90% or at least about 95% sequence identity.

Homologs of the disclosed nucleic acid sequences are typicallycharacterized by possession of at least 40% sequence identity countedover the full length alignment with the amino acid sequence of thedisclosed sequence using the NCBI Blast 2.0, gapped blastn set todefault parameters. A preferred method utilizes the BLASTN module ofWU-BLAST-2 (Altschul et al., 1996); set to the default parameters, withoverlap span and overlap fraction set to 1 and 0.125, respectively.Nucleic acid sequences with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 90% orat least about 95% sequence identity.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequences in thefigures, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than that shown in thefigures as discussed below, will be determined using the number of aminoacids in the longer sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedherein for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

As will be appreciated by those skilled in the art, the sequences of thepresent invention may contain sequencing errors. That is, there may beincorrect nucleosides, frameshifts, unknown nucleosides, or other typesof sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include one or morenucleic acid sequences that permit it to replicate in one or more hostcells, such as origin(s) of replication. A vector may also include oneor more selectable marker genes and other genetic elements known in theart.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, plant or animalcell, including transfection with viral vectors, transformation byAgrobacterium, with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration and includestransient as well as stable transformants.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell or the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term embracesnucleic acids including chemically synthesized nucleic acids and alsoembraces proteins prepared by recombinant expression in vitro or in ahost cell and recombinant nucleic acids as defined below.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary, join two protein-coding regions in the samereading frame. With respect to polypeptides, two polypeptide sequencesmay be operably linked by covalent linkage, such as through peptidebonds or disulfide bonds.

Recombinant: By “recombinant nucleic acid” herein is meant a nucleicacid that has a sequence that is not naturally occurring or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination is oftenaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of of nucleic acids, e.g., by genetic engineeringtechniques, such as by the manipulation of at least one nucleic acid bya restriction enzyme, ligase, recombinase, and/or a polymerase. Onceintroduced into a host cell, a recombinant nucleic acid is replicated bythe host cell, however, the recombinant nucleic acid once replicated inthe cell remains a recombinant nucleic acid for purposes of thisinvention. By “recombinant protein” herein is meant a protein producedby a method employing a recombinant nucleic acid. As outlined above“recombinant nucleic acids” and “recombinant proteins” also are“isolated”, as described above.

Complementary DNA (cDNA): A piece of DNA that is synthesized in thelaboratory by reverse transcription of an RNA, preferably an RNAextracted from cells. cDNA produced from mRNA typically lacks internal,non-coding segments (introns) and regulatory sequences that determinetranscription.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Transgenic plant: As used herein, this term refers to a plant thatcontains recombinant genetic material not normally found in plants ofthis type and which has been introduced into the plant in question (orinto progenitors of the plant) by human manipulation. Thus, a plant thatis grown from a plant cell into which recombinant DNA is introduced bytransformation is a transgenic plant, as are all offspring of that plantthat contain the introduced transgene (whether produced sexually orasexually). It is understood that the term transgenic plant encompassesthe entire plant and parts of said plant, for instance grains, seeds,flowers, leaves, roots, fruit, pollen, stems etc.

The present invention is applicable to both dicotyledonous plants (e.g.tomato, potato, soybean, cotton, tobacco, etc.) and monocotyledonousplants, including, but not limited to graminaceous monocots such aswheat (Triticum spp.), rice (Oryza spp.), barley (Hordeum spp.), oat(Avena spp.), rye (Secale spp.), corn (Zea mays), sorghum (Sorghum spp.)and millet (Pennisetum spp). For example, the present invention can beemployed with barley genotypes including, but not limited to Morex,Harrington , Crystal, Stander, Moravian III, Galena, Salome, Steptoe,Klages, Baronesse, and with wheat genotypes including, but not limitedto Yecora Rojo, Bobwhite, Karl and Anza. In general, the invention isparticularly useful in cereals.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified barleythioredoxin h protein preparation is one in which the barley thioredoxinh protein is more enriched or more biochemically active or more easilydetected than the protein is in its natural environment within a cell orplant tissue. Accordingly, “purified” embraces or includes the removalor inactivation of an inhibitor of a molecule of interest. In apreferred embodiment, a preparation of barley thioredoxin h protein ispurified such that the barley thioredoxin h represents at least 5-10% ofthe total protein content of the preparation. For particularapplications, higher protein purity may be desired, such thatpreparations in which barley thioredoxin h represents at least 50% or atleast 75% or at least 90% of the total protein content may be employed.

Ortholog: Two nucleotide or amino acid sequences are orthologs of eachother if they share a common ancestral sequence and diverged when aspecies carrying that ancestral sequence split into two species,sub-species, or cultivars. Orthologous sequences are also homologoussequences.

II. Production of Plants With Elevated Seed Thioredoxin

Standard molecular biology methods and plant transformation techniquescan be used to produce transgenic plants that produce seeds having anelevated level of thioredoxin protein. The following sections providegeneral guidance as to the selection of particular constructs andtransformation procedures.

a. Constructs

The present invention utilizes recombinant constructs that are suitablefor obtaining elevated expression of thioredoxin in plant seeds relativeto non-transformed plant seeds. In their most basic form, theseconstructs may be represented as P-T, wherein P is a seed-specificpromoter and T is a nucleic acid sequence encoding thioredoxin. Inanother embodiment, a peptide signal sequence that targets expression ofthe thioredoxin polypeptide to an intracellular body may be employed.Such constructs may be represented as P-SS-T, wherein SS is the signalpeptide. Nucleic acid molecules that may be used as the source of eachof these components are described in the Definitions section above.

Each component is operably linked to the next. For example, where theconstruct comprises the hordein D-promoter (P), the hordein D-signalsequence (SS) encoding the hordein signal peptide, and an open readingframe encoding, preferably, the wheat thioredoxin h protein (T), thehordein promoter is linked to the 5′ end of the sequence encoding thehordein signal sequence, and the hordein signal sequence is operablylinked to the 5′ end of the thioredoxin open reading frame, such that Cterminus of the signal peptide is joined to the N-terminus of theencoded protein.

The construct will also typically include a transcriptional terminationregion following the 3′ end of the encoded protein ORF. Illustrativetranscriptional termination regions include the nos terminator fromAgrobacterium Ti plasmid and the rice alpha-amylase terminator.

Standard molecular biology methods, such as the polymerase chainreaction, restriction enzyme digestion, and/or ligation may be employedto produce these constructs comprising any nucleic acid molecule orsequence encoding a thioredoxin protein or polypeptide.

b. General Principles of Plant Transformation

Introduction of the selected construct into plants is typically achievedusing standard transformation techniques. The basic approach is to: (a)clone the construct into a transformation vector; which (b) is thenintroduced into plant cells by one of a number of techniques (e.g.,electroporation, microparticle bombardment, Agrobacterium infection);(c) identify the transformed plant cells; (d) regenerate whole plantsfrom the identified plant cells, and (d) select progeny plantscontaining the introduced construct. Preferably all or part of thetransformation vector will stably integrate into the genome of the plantcell. That part of the transformation vector which integrates into theplant cell and which contains the introduced P-T or P-SS-T sequence (theintroduced “thioredoxin transgene”) may be referred to as therecombinant expression cassette.

Selection of progeny plants containing the introduced transgene may bemade based upon the detection of thioredoxin or NTR over-expression inseeds, or upon enhanced resistance to a chemical agent (such as anantibiotic) as a result of the inclusion of a dominant selectable markergene incorporated into the transformation vector.

Successful examples of the modification of plant characteristics bytransformation with cloned nucleic acid sequences are replete in thetechnical and scientific literature. Selected examples, which serve toillustrate the knowledge in this field of technology include:

-   U.S. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and Methods”);-   U.S. Pat. No. 5,677,175 (“Plant Pathogen Induced Proteins”);-   U.S. Pat. No. 5,510,471 (“Chimeric Gene for the Transformation of    Plants”);-   U.S. Pat. No. 5,750,386 (“Pathogen-Resistant Transgenic Plants”);-   U.S. Pat. No. 5,597,945 (“Plants Genetically Enhanced for Disease    Resistance”);-   U.S. Pat. No. 5,589,615 (“Process for the Production of Transgenic    Plants with Increased Nutritional Value Via the Expression of    Modified 2S Storage Albumins”);-   U.S. Pat. No. 5,750,871 (“Transformation and Foreign Gene Expression    in Brassica Species”);-   U.S. Pat. No. 5,268,526 (“Overexpression of Phytochrome in    Transgenic Plants”);-   U.S. Pat. No. 5,780,708 (“Fertile Transgenic Com Plants”);-   U.S. Pat. No. 5,538,880 (“Method For Preparing Fertile Transgenic    Corn Plants”);-   U.S. Pat. No. 5,773,269 (“Fertile Transgenic Oat Plants”);-   U.S. Pat. No. 5,736,369 (“Method For Producing Transgenic Cereal    Plants”);-   U.S. Pat. No. 5,610,049 (“Methods For Stable Transformation of    Wheat”).

These examples include descriptions of transformation vector selection,transformation techniques and the construction of constructs designed toexpress an introduced transgene.

c. Plant Types

The transgene-expressing constructs of the present invention may beusefully expressed in a wide range of higher plants to obtain seed- orgrain-specific expression of selected polypeptides. The invention isexpected to be particularly applicable to monocotyledonous cereal plantsincluding barley, wheat, rice, rye, maize, triticale, millet, sorghum,oat, forage, and turf grasses. In particular, the transformation methodsdescribed herein will enable the invention to be used with genotypes ofbarley including Morex, Harrington, Crystal, Stander, Moravian III,Galena, Golden Promise, Steptoe, Klages and Baronesse, and commerciallyimportant wheat genotypes including Yecora Rojo, Bobwhite, Karl andAnza.

The invention may also be applied to dicotyledenous plants, including,but not limited to, soybean, sugar beet, cotton, beans, rape/canola,alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut,clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato,carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brusselssprouts, peppers; and tree fruits such as citrus, apples, pears,peaches, apricots, and walnuts.

d. Vector Construction

A number of recombinant vectors suitable for stable transformation ofplant cells or for the establishment of transgenic plants have beendescribed including those described in Weissbach and Weissbach, (1989),and Gelvin et al., (1990). Typically, plant transformation vectorsinclude one or more ORFs under the transcriptional control of 5′ and 3′regulatory sequences and a dominant selectable marker with 5′ and 3′regulatory sequences. The selection of suitable 5′ and 3′ regulatorysequences for constructs of the present invention is discussed above.Dominant selectable marker genes that allow for the ready selection oftransformants include those encoding antibiotic resistance genes (e.g.,resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin orspectinomycin) and herbicide resistance genes (e.g, phosphinothricinacetyltransferase).

e. Transformation and Regeneration Techniques

Methods for the transformation and regeneration of both monocotyledonousand dicotyledonous plant cells are known, and the appropriatetransformation technique will be determined by the practitioner. Thechoice of method will vary with the type of plant to be transformed;those skilled in the art will recognize the suitability of particularmethods for given plant types. Suitable methods may include, but are notlimited to: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG) mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium mediated transformation. Typical procedures fortransforming and regenerating plants are described in the patentdocuments listed at the beginning of this section.

f. Selection of Transformed Plants

Following transformation, transformants are preferably selected using adominant selectable marker. Typically, such a marker will conferantibiotic or herbicide resistance on the seedlings of transformedplants, and selection of transformants can be accomplished by exposingthe seedlings to appropriate concentrations of the antibiotic orherbicide. After transformed plants are selected and grown to maturityto allow seed set, the seeds can be harvested and assayed forover-expression of thioredoxin.

III. Use of Plants, Seeds or Grains Expressing Elevated Levels ofThioredoxin

In one embodiment, the transgene protein, for example thioredoxinexpressed in plants, especially seeds or grains, using the methodsdescribed herein, is used in the production and synthesis ofthioredoxin. The thioredoxin transgene expressed by the recombinantnucleic acid of the invention may be harvested at any point afterexpression of the protein has commenced. When harvesting from the seedor grain or other part of a plant for example, it is not necessary forthe seed or grain or other part of the plant to have undergonematuration prior to harvesting. For example, transgene expression mayoccur prior to seed or grain maturation or may reach optimal levelsprior to seed or grain maturation. The transgene protein may be isolatedfrom the seeds or grain, if desired, by conventional proteinpurification methods. For example, the seed or grain can be milled, thenextracted with an aqueous or organic extraction medium, followed bypurification of the extracted thioredoxin protein. Alternatively,depending on the nature of the intended use, the transgene protein maybe partially purified, or the seed or grain may be used directly withoutpurification of the transgene protein for food processing or otherpurposes.

For example, the addition of thioredoxin promotes the formation of aprotein network that produces flour with enhanced baking quality.Kobrehel et al., (1994) have shown that the addition of thioredoxin toflour of non-glutenous cereal such as rice, maize, and sorghum promotesthe formation of a dough-like product. Accordingly, the addition ofthioredoxin expressed in seeds using the methods described herein finduse in the production of flour with improved baking quality such asincreased strength and/or volume.

The enhanced expression of thioredoxin also produces a seed having analtered biochemical composition. For example, enhanced thioredoxinexpression produces seed with increased enzymatic activity, such as,increased pullulanase and alpha-amylase A. Enhanced thioredoxinexpression also produces seed with early alpha-amylase B activation.Pullulanase (“debranching enzyme”) is an enzyme that breaks downbranched starch of the endosperm of cereal seeds by hydrolyticallycleaving alpha-1,6 bonds. Alpha-amylases break down starch 1-4 linkages.Pullulanase and amylases are enzymes fundamental to the brewing andbaking industries. Pullulanase and amylases are required to break downstarch in malting and in certain baking procedures carried out in theabsence of added sugars or other carbohydrates. Obtaining adequateactivity of these enzymes is problematic especially in the maltingindustry. It has been known for some time that dithiothreitol (DTT, achemical reductant that reduces and sometimes replaces thioredoxin)activates pullulanase of cereal preparations (e.g., barley, oat, andrice flours). A method of adequately increasing the activity ofpullulanase and alpha-amylase A and shortening the activation time ofalpha-amylase B with a physiologically acceptable system, leads to morerapid malting methods and, owing to increased sugar availability, toalcoholic beverages such as beers-with reduced carbohydrate content.

Accordingly, seeds or grains with enhanced thioredoxin expressionprovide advantages in the production of malt and beverages produced by afermentation process. Enhanced pullulanase and alpha-amylase A andearlier induction of alpha-amylase B in grain increases the speed andefficiency of germination, important in malting, where malt is producedhaving increased enzymatic activity resulting in enhanced hydrolysis ofstarch to fermentable carbohydrates, thereby, improving the efficiencyof fermentation in the production of alcoholic beverages, for example,beer and scotch whiskey. Early alpha-amylase B activation would reducethe total time for malting by about 20%. Enhanced fermentation processesalso find use in the production of alcohols that are not intended forhuman consumption, i.e., industrial alcohols.

In another embodiment, seed or grains with enhanced thioredoxinexpression provide advantages in enhancing the onset and efficiency ofgermination.

The overexpression of thioredoxin in seed or grains results in anincrease in the total protein. It also promotes the redistribution ofproteins to the most soluble albumin/globulin fraction and theproduction of flour and other food products, feed, and beverages withimproved digestibility in comparison to edible products made fromnon-transformed grains. Such edible products find use in ameliorationand treatment of food malabsorptive syndromes, for example, sprue orcatarrhal dysentery. Sprue is a malabsorptive syndrome affecting bothchildren and adults, precipitated by the ingestion of gluten-containingfoods. Edible products that are more readily digested and readilyabsorbed avoid or ameliorate the disease symptoms. Edible products withimproved digestibility also ameliorate or reduce symptoms associatedwith celiac disease in which storage proteins that are not readilydigested in afflicated individuals result in inflammation of the GItract.

The expression of thioredoxin in seed grains results in the productionof foods and other edible products with reduced allergenicity incomparison to edible products made from non-transformed grains. Foodallergies are a significant health and nutrition problem (Lehrer et al.,1996). Up to 2% of adults and 8% of children have a food allergy causingserious symptoms including death. Wheat protein is one of the principalallergens. Food allergies are defined by the American academy of Allergyand Immunology Committee on Adverse Reactions to Food as “animmunological reaction resulting from the ingestion of a food or a foodadditive” (Fenema, 1996; Lasztity, 1996). Most true allergic responsesto food proteins appear to be caused by a type-I imunolobulin E(IgE)-mediated hypersensitivity reaction (Sicherer, 1999). Theseresponses may occur within minutes or a few hours after eating theoffending food (Furlong-Munoz, 1996). When the offending food isinjested by allergy-sensitive individuals the body releases histaminesand other biochemicals, resulting in itchy eyes, rash or hives; runnynose; swelling of the lips, tongue, and face; itching or tightness ofthe throat; abdominal pain; nausea; diarrhea; and shortness of breath.Some individuals have severe, anaphylactic reactions, resulting inapproximately 135 deaths per year in the United States. In the U.S. over2,500 emergency rooms visits per year are allergy-related. There is nocure for food allergies, only avoidance of the food will preventsymptoms. For example, patients with wheat allergy must avoid wheat- orgluten-containing foods; wheat gluten is a very common ingredient inmany processed foods (Marx et al., 1999).

A feature common to many allergens is the presence of one or moredisulfide bonds that contribute to the resistance of allergens todigestion (Astwood et al., 1996), allowing them to be mostly intact whenthey react the small intestine where they are presented to mucosal cellsthat mount an IgE immune response. The major allergens were found to beinsoluble storage proteins, gliadins and glutenins. The soluble storageproteins, albumins and globulins were considerably weaker (Buchanan etal., 1997). Allergenicity of these proteins is substantially decreasedafter thioredoxin treatment and disulfide bond reduction.

Edible products, for example, bread, cookies, dough, thickeners,beverages, malt, pasta, food additives, including animal feeds, madeusing the transgenic plants or parts of a transgenic plant of theinvention have decreased allergenicity and accordingly can be used to inthe treatment of an allergic response. By “treatment” or “alleviating”symptoms herein is meant prevention or decreasing the probability ofsymptoms.

Increased digestibility of seeds or grains also provides widerconsumption of grains by man and animals who otherwise can not consumesuch grains. For example, sorghum is the world's fifth leading grain interms of metric tons after wheat, rice, maize, and barley and third inproduction in the Untied States after maize and wheat. The use ofsorghum is constrained in part because of the difficulty associated withthe digestibility of its protein and starch compared to other grains.This difficulty with the digestibility of sorghum protein and starch hasto do with the structure of the seed and the manner in which theproteins are associated with the starch. The digestibility of the starchflour from sorghum cultivars is 15-25% lower in digestibility than, forexample, maize. Perhaps more notable is the fact that, unlike othergrains, the indigestibility of unprocessed sorghum flour increasesdramatically after boiling in water, a common practice in Africa. Astudy with human subjects showed that protein digestibility in cookedsorghum porridge can be as low as 46%, whereas the percent digestibilityfor cooked wheat, maize, and rice was 81%, 73%, and 66% respectively(Mertz et al. 1984, MacLean et al. 1981). Exogenous addition of reducingagents increases the digestibility of the starch (Hamaker et al. 1987).However, the efficacy of manipulating the thioredoxin system in vivo inthe seed by expressing increased amounts of thioredoxin in a mannerwhich does not adversely affect plant development or morphology had notpreviously been demonstrated. Accordingly, the transgenic plants of theinvention provide wider use of seeds or grains as food sources byincreasing the digestibility of the starch and/or protein component. Thetransgenic seeds or grains of the present invention also provide theadvantage of increasing the digestibility of food products for human andfeed for animals made of these grains without the addition of exogenousreducing agents. In addition, the increased digestibility results ingreater utilization of the food or feed, i.e., a human or animalconsuming an edible product comprising a transgenic seed or grain of theinvention or an extract thereof more efficiently absorbs nutrients andtherefore requires to consume less in comparison to a non-transgenicfood product. In another embodiment the transgenic seed, grain orextracts thereof of the present invention and extracts or food productsthereof are used as a food or feed additives. For example, an extract orflour or malt produced from a transgenic seed or grain of the inventionis added to a non-transgenic food or feed product to improve thedigestibility or decrease the allergenicity of the nontransgenic foodproduct or to improve the quality of the total food product, such as, byincreasing the strength and/or volume of the food product.

Illustrative embodiments of the invention are described below,

EXAMPLES Example 1 Expression of Wheat Thioredoxin h (WTRXh) inTransgenic Barley

Four different DNA constructs were produced, each containing a 384-bpwtrxh fragment encoding the 13.5-KDa WTRXh protein. The four constructsare illustrated in FIG. 1 and described below. Each construct comprisedthe 384-bp wtrxh fragment operably linked to a seed-specific promoter(either the barley endosperm-specific D-hordein or B1-hordein promotersor the maize embryo-specific globulin promoter). An additional constructcomprised the 384-bp wtrxh fragment operably linked to the B1-hordeinpromoter and the B1-hordein signal sequence (FIG. 6). The transformationvector used included the bar gene, conferring resistance to bialaphos.Twenty-eight independent regenerable barley lines were obtained afterbialaphos selection and all were PCR-positive for the bar gene. Thepresence of the wtrxh gene was confirmed in the genome of the 28independent lines by PCR and DNA hybridization analyses. The expressionof the WTRXh protein was assessed by western blot analysis, usingpurified wheat thioredoxin as a control. The WTRXh expressed intransgenic barley had a molecular mass that differed from native barleyTRXh but was identical to WTRXh. The WTRXh was found to be highlyexpressed in developing and mature seed of transgenic barley plantsalthough levels of expression varied among the transgenic events. Onaverage, higher expression levels were observed in lines transformedwith the DNA construct containing the B1-hordein promoter plus thesignal peptide sequence than the same promoter without the signalpeptide sequence. The WTRXh purified from transgenic barley seed wasconfirmed to be biochemically active.

A. Materials and Methods

Plant Materials for Transformation

A two-rowed spring cultivar of barley, Golden Promise, was grown ingrowth chambers as described previously (Wan and Lemaux 1994; Lemaux etal., 1996).

Construction of Wheat Thioredoxin h Expression Vectors and DNASequencing

Expression vectors were constructed containing the wheat thioredoxin hgene (wtrxh) driven by the barley endosperm-specific B1- or D-hordeinpromoter or the maize embryo-specific globulin promoter.

The plasmids were constructed as follows.

(1) pDhWTRXN-2: A 384-bp wtrxh coding region was amplified by PCR frompTaM13.38 (Gautier et al., 1998). This plasmid contained a cDNA ofwtrxh, which was used as a template, creating XbaI and SacI sites withthe following primers Wtrxh1 (5′-atatctagaATGGCGGCGTCGGCGGCGA) (SEQ IDNO:5) and Wtrxh2R (5′-atagagctcTTACTGGGCCGCGTG TAG) (SEQ ID NO:6),respectively (FIG. 1). Small letters in the primer denote a restrictionenzyme site for subcloning of the DNA fragment containing the wtrxhgene; underlined letters denote wtrxh sequences. The ATG initiationcodon for wtrxh expression was included in the Wtrxh1 primer. PCRreactions were performed on a thermocycler (MJ Research Inc., Watertown,Mass.) using recombinant Taq DNA polymerase (Promega. Madison, Wis.) ina 100-μl reaction volume. The reaction buffer contained 10 mM Tris-HCl(pH 9.0), 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton-X-100, and 50 μM of eachdeoxyribonucleoside triphosphate. PCR conditions utilized 25 cycles of94° C. for 1 min, 55° C. for 1 min and 72° C. for 2 min, with a finalextension step at 72° C. for 7 min. The wtrxh fragment, which wasamplified with the primers Wtrxh1 and Wtrxh2R, was purified from a 0.7%agarose gel using a QIAquick® gel extraction kit (Qiagen Inc.,Chatsworth, Calif.), digested with XbaI and SacI and ligated intoXbaI/SacI-digested pUC19 to generate the pWTRXh-1 plasmid. Nucleotidesequences of the PCR-amplified wtrxh coding region fragment weredetermined by the dideoxynucleotide chain termination method usingSequenase according to manufacturer's instructions (United StatesBiochemical, Cleveland, Ohio) with double-stranded plasmid templates andregularly spaced primers

pDhWTRXN-2 was made by replacing the uidA gene in pDhGN-2 (containingbarley endosperrn-specific D-hordein promoter (FIG. 7) and nos 3′terminator) with the XbaI/SacI fragment containing the wtrxh codingsequence from pWTRXh-I, which contains the PCR-amplified wtrxh codingsequence in pUC19. To construct pDhGN-2 a 0.4-kb D-hordein promoter wasamplified by PCR from pDII-Hor3 (Sørenson et al., 1996; Cho et al.,1999a). This plasmid contained the D-hordein promoter sequence, whichwas used as a template, creating Sphl and XbaI sites with the followingprimers: Dhor1 (5′-ggcgcatgcgaattcGAATTCGATATCGATCTTCGA-3′) (SEQ IDNO:23) and Dhor2 (5′-aactctagaCTCGGTGGACTGTCAATG-3′) (SEQ ID NO:12),respectively. Small letters in the primers contain restriction enzymesites for subcloning of the DNA fragment containing the D-hordeinpromtoer; underlined letters denote D-hordein promoter sequences. ThePCR amplified D-hordein promoter fragment was digested with Sphl andXbaI and repalced with the cauliflower mosaic 35S (CaMV 35S) promoter inp35SGN-3 to generate the pDhGN-2 plasmid. p35SGN-3 was made by ligatingthe 3.0-kb Sphl-EcoRl fragment containing the CaMV 35S promoter, uidA(beta-glucuronidase, gus) gene and nos into the Sphl/EcoRl-digestedpUC18.

(2) pdBhWTRX-1: The construction of pdBhWTRXN-1 started by usingpBhWTRXN-1. pBhWTRXN-1 was made by replacing the uidA gene in pBhGN-1,which contains uidA driven by the barley endosperm-specific B1-hordeinpromoter and terminated by the nos 3′ terminator, with the XbaI/SacIfragment from pWTRXh-1, which contains the wtrxh coding sequence. The120-bp HindIII-5′ B1-hordein flanking region was deleted from thepBhWTRXN-1 and religated to make the pdBhWTRXN-1 construct.

(3) pdBhssWTRXN3-8: Primers Bhor7 (5′-GTAAAGCITTAACAACCCACACATTG) (SEQID NO:7) and BhorWtrxh1R (5′-CCGACGCCGCTGCAATCGTACTTGTTGCCGCAAT) (SEQ IDNO:8) containing HindIII and Acyl sites, respectively, were used foramplification of a 0.49-kb B1-hordein 5′-region, which included theB1-hordein signal peptide sequence (FIG. 6). A λ2-4/HindIII plasmidcontaining a genomic clone of B1-hordein (Brandt et al., 1985; Cho andLemaux, 1997) was used as a template for the amplification. The primerBhorWtrxh1R is an overlapping primer, which contains the wtrxh codingsequence (underlined) and a partial signal peptide sequence from theB1-hordein promoter, but lacks the ATG initiation codon for wtrxh.pdBhssWTRXN3-8 was made by replacing the D-hordein promoter (FIG. 7) inpDhWTRXN-2 with the 0.49-kb PCR-amplified HindIII/Acyl fragment, whichcontains the B1-hordein promoter, its signal peptide sequence and thejunction region from the 5′ trxh gene. Thus, construct pdBhssWTRXN3-8contains the barley endosperm-specific B1-hordein promoter with itssignal peptide sequence (FIG. 6), wtrxh , and nos (FIG. 1). The signalpeptide sequence containing the ATG initiation codon was directlycombined with the sequence of wtrxh, with no extra amino acid sequencesbeing introduced between the two. This ensures that the WTRXh proteinhas a precise cleavage site in the lumen of the endoplasmic reticulum(ER). The authenticity of a PCR-amplified fragment from the chimericproduct was confirmed by DNA sequencing.

(4) pGlb1WTRXN-1: The 1.42-kb HindIII/BamHI fragment containing themaize embryo-specific globulin promoter from the ppGlb1GUS plasmid (Liuand Kriz, 1996) was ligated into pBluescript II KS(+) to create HindIIIand XbaI sites. pGlbWTRXN-1 was made by restricting pDhWTRXN-2 withHindIII and XbaI in order to remove the 0.49-kb HindIII/XbaI barleyD-hordein promoter from the pDhWTRXN-2. In place of the 0.49-kbHindIII/XbaI D-hordein promoter fragment (FIG. 7), the 1.42-kbHindIII/,XbaI maize globulin promoter was ligated into the HindIII/XbaIdigested pDhWTRXN-2 to form the pGlbWTRXN-1 plasmid.

Stable Barley Transformation

Stable transgenic lines of barley expressing WTRXh driven by theB1-hordein promoter with and without the signal peptide sequence (FIG.6), by the D-hordein promoter (FIG. 7) and by the maize globulinpromoter were obtained following modifications of published protocols(Wan and Lemaux 1994; Lemaux et al., 1996; Cho et al., 1998a-c). Wholeimmature embryos (IEs) (1.0-2.5 mm) were aseptically removed, placedscutellum-side down on DC callus-induction medium containing 2.5 mg/L2,4-D and 5 μM CuSO₄ (Cho et al., 1998a-c). One day after incubation at24±1° C. in the dark, the IEs were transferred scutellum-side up to DCmedium containing equimolar amounts of mannitol and sorbitol to give afinal concentration of 0.4 M. Four hours after treatment with theosmoticum, the IEs were used for bombardment. Gold particles (1.0 μm)were coated with 25 μg of a 1:1 molar ratio of pAHC20 (Christensen andQuail, 1996) and one of the following plasmids, pdBhWTRXN-1,pdBhssWTRXN3-8, pDhWTRXN-2 and pG1bWTRXN-1. The microprojectiles werebombarded using a PDS-1000 He biolistic device (Bio-Rad, Inc., Hercules,Calif.) at 1100 psi. Bombarded IEs were selected on DC medium with 5mg/L bialaphos for 2 to 3 months. Bialaphos-resistant callus wastransferred onto an intermediate culturing medium (DBC2; Cho et al.,1998a-c), containing 2.5 mg/L 2,4-D, 0.1 mg/L BAP and 5.0 μM CuSO₄,between the selection [DC medium plus bialaphos (Meiji Seika Kaisha,Ltd., Yokohama, Japan)] and regeneration (FHG medium; Hunter, 1988)steps. The culturing after callus induction and selection on DC mediumwere carried out under dim light conditions (approximately 10 to 30 μE,16 h-light) (Cho et al., 1998a-c). Regenerated shoots were transferredto Magenta boxes containing rooting medium (callus-induction mediumwithout phytohormones) containing 3 mg/L bialaphos. When shoots reachedthe top of the box, plantlets were transferred to soil in thegreenhouse.

Cytological Analysis

For cytological analysis of transgenic barley plants. healthy rootmeristems were collected from young plants grown in the greenhouse.After pre-treatment at 4° C. in saturated 1-bromonaphthalene solutionovernight, root meristems were fixed in 1:3 glacial acetic acid:ethanoland stored at 4° C. Root meristems were hydrolyzed in 1 M HCl at 60° C.for 5-7 min, stained in Feulgen solution and squashed on a glass slidein a drop of 1% aceto-carmine. Chromosomes were counted from at leastfive well-spread cells per plant.

Herbicide Application

To determine herbicide sensitivity of T₀ plants and their progeny, asection of leaf blade at the 4- to 5-leaf stage was painted using acotton swab with 0.25% (v/v) Basta™ solution (starting concentration 200g/L phophinothricin, Hoechst AG, Frankfurt, Germany) plus 0.1 % Tween20. Plants were scored 1 week after herbicide application.

Polymerase Chain Reaction (PCR) and DNA Blot Hybridization

Total genomic DNA from leaf tissues was purified as described byDellaporta (1993). To test for the presence of wtrxh in genomic DNA ofputatively transformed lines, 250 ng of genomic DNA was amplified by PCRusing one of two primer sets: Set 1: Wtrxh1(5′-ATATCTAGAATGGCGGCGTCGGCGGCGA) (SEQ ID NO:5) and Wtrxh2R(5′-ATAGAGCTCTTACTGGGCCGCGTGTAG); (SEQ ID NO:6) or Set 2: Wtrxh4(5′-CCAAGAAGTTCCCAGCTGC) (SEQ ID NO:11) and Wtrxh5R(5′-ATAGCTGCGACAACCCTGTCCTT). (SEQ ID NO:19)

The presence of bar was determined using the primer set: BAR5F(5′-CATCGAGACAAGCACGGTCAACTTC3′) (SEQ ID NO:13) and BAR1R(5′-ATATCCGAGCGCCTCGTGCATGCG) (SEQ ID NO:14) (Lemaux et al., 1996).Amplifications were performed with Taq DNA polymerase (Promega, Madison,Wis.) in a 25-μl reaction (Cho et al., 1998a-c). Twenty-five microlitersof the PCR product with loading dye were subjected to electrophoresis ina 1.0% agarose gel with ethidium bromide and photographed using exposureto UV light. Presence of 0.4- and 0.14-kb fragments was consistent withintact and truncated wtrxh fragments, respectively; an internal 0.34-kbfragment was produced from the bar gene with bar primers. Homozygouslines for wtrxh were screened by PCR and western blot analysis in T₂ orT₃ plants.

For DNA hybridization analysis, 10 μg of total genomic DNA from leaftissue of each line was digested with HindIII and SacI, separated on a1.0% agarose gel, transferred to Zeta-Probe GT membrane (Bio-Rad,Hercules, Calif.) and hybridized with a radiolabeled wtrxh-specificprobe following the manufacturer's instructions. The wtrxh-containing0.4 kb XbaI-SacI fragment from pDhWTRXN-9 was purified by QIAEX gelextraction kit (QIAGEN, Chatsworth, Calif.) and labeled with ³²P-dCTPusing random primers

Western Blot Analysis

Western blot analysis was performed on seeds from selected transgeniclines as well as from control barley seeds from non-transgenic GoldenPromise grown under the same conditions as the transgenic plants andfrom control wheat seeds of a durum wheat cultivar, cv. Monroe, or abread wheat cultivar cv. Capitale. Whole seeds were ground to a finepowder with a mortar and pestle under liquid nitrogen. Ten to 20 seedswere used for each sample; the volume of extraction buffer (50 mM TrisHCl or phosphate buffer, pH 7.8, 0.5 mM phenylmethyl sulfonyl fluoride[PMSF], 1 mM EDTA) varied from 2 to 4 ml depending on the number ofseeds used and the viscosity of the extract. Grinding was continued foran additional minute after buffer addition; the mixture was thencentrifuged at 14,000×g for 10 minutes and the supernatant solution wassaved as the albumin-globulin fraction that contained the thioredoxin.

SDS-PAGE of the albumin-globulin fraction was performed in 12-17%polyacrylamide gradient gels at pH 8.5 (Laemmli, 1970). From each sampleequal amounts of protein (˜40 μg) quantitated according to Bradford(1976) were diluted 1:2 v/v in Laemmli sample buffer, boiled for 3minutes, loaded onto gels and subjected to electrophoresis at a constantcurrent of 15 mA. Proteins were transferred to nitrocellulose at aconstant voltage of 40 V for 4 hours at 4° C. using a Hoefer TransphorTransfer Unit (Alameda, Calif.). Nitrocellulose was blocked with 5%powdered milk in TBS for 2 hours at room temperature (RT), incubated inprimary antibody for 4 hours at RT and in secondary antibody for 1 hourat RT. Primary antibody was wheat anti-thioredoxin h II Ab (Johnson etal., 1987b) diluted 1 to 500; secondary antibody was goat anti-rabbitalkaline phosphatase (Bio-Rad, Hercules Calif.) diluted 1:3000. Blotswere developed in NBT/BCIP alkaline phosphatase color reagent (accordingto Bio-Rad instructions); gels were stained with Coomassie blue toassure transfer. Images were scanned using a Bio-Rad GelDoc 1000(Hercules, Calif.) and analyzed using Bio-Rad Multi Analyst, version1.0.2. All bands were scanned over the same area, using a rectangle ofcomparable density as background; results were expressed as % of volumescanned. The number shown represents the percent of the total volume(pixel density×area of scanned band).

WTRXh Activity Measurements

Preparation of Materials for Extraction.

Mature grains from various heterozygous and homozygous transgenic linesserved as starting materials for the assay. Heterozygous lines with aD-hordein promoter were: GPDhBarWtrx-5, GPDhBarWtrx-9-1, andGPDhBarWtrx-9-2. Heterozygous lines with a B-hordein promoter and nosignal sequence were: GPdBhBarWtrx-2, -5, -9, -19 and GPdBhBarWtrx-20.Heterozygous lines with a B-hordein promoter plus a signal sequencewere: GPdBhssBarWtrx-2, -7, GPdBhssBarWtrx-29, GPdBhssBarWtrx-20,GPdBhssBarWtrx-14, GPdBhssBarWtrx-22. Homozygous lines with a signalsequence were: GPdBhssBarWtrx-2-17, GPdBhssBarWtrx-2-17-1,GPdBhssBarWtrx-29-3 and GPdBhssBarWtrx-29-3-2. Control materialsincluded a non-transformed tissue culture derived line, 4-96, atransformed line containing only bar, GPBar-l, and null segregant lines,GPdBhssBarWtrx-29-11 and GPdBhssBarWtrx-29-11-10, derived from lineGPdBhssBarWtrx-29.

Preparation of (NH₄)₂SO₄ Extracts for Gel Filtration

Approximately fifteen grams of barley grains were ground to powder in acoffee grinder and extracted with 80 ml (1:4 w/v) of buffer [(50 mMTris-HCl buffer, pH 7.9, 1 mM EDTA, 0.5 mM PMSF (phenylmethysulfonylfluoride)], 2 mM e-amino-n caproic acid, 2 mM benzamidine-HCl) bystirring for 3 hrs at 4° C. The slurry plus the rinse was subjected tocentrifugation at 25,400×g for 20 min, the supernatant solution wasdecanted through glass wool, pellets were resuspended in a small volumeof buffer and then clarified by centrifugation as before. Thesupernatant fractions were combined, an aliquot was removed and theremainder was subjected to acidification by adjusting the pH from 7.83to 4.80 with 2 N formic acid; denatured proteins were removed bycentrifugation as above prior to assay. The pH of the acidifiedsupernatant solution was readjusted to 7.91 with 2 N NH₄OH and analiquot was removed for assay. Powdered (NH₄)₂SO₄ was added to a finalconcentration of 30% and the sample was stirred for 20 min at 4° C.,followed by centrifugation as described above. The pellet was discarded.Additional (NH₄)₂SO₄ was added to bring the decanted supernatantsolution to 90% saturation; the sample was stirred for 16 hrs at 4° C.,followed by centrifugation as described above.

The supernatant solution was discarded, the 30-90% (NH₄)₂SO₄ pelletswere re-suspended in 30 mM Tris-HCl, pH 7.9 buffer and then subjected tocentrifugation at 40,000×g for 15 min to clarify. The resultingsupernatant (30-90% (NH₄)₂SO₄ fraction) was added to dialysis tubing(6,000-8,000 MW cut-off) and exposed to solid sucrose at 4° C. to obtaina 10-fold reduction in volume. An aliquot (1 ml) of the clarified andconcentrated 30-90% (NH₄)₂SO₄) sample was saved and the remaining samplewas applied to a pre-equilibrated (30 mM Tris-HCl, pH 7.9, 200 mM NaCl)Sephadex G-50 superfine column (2.5×90 cm;˜400 mL bed volume) with aperistaltic pump at a flow rate of 0.5 mL/min. Protein was eluted withthe same buffer at the same flow rate; one hundred fifty drop-fractionswere collected. Selected fractions were used to measure absorbance at280 nm using a Pharmacia Biotech Ultrospec 4000 and to assay for TRXhactivity following the NADP-MDH activation protocol (see below). Activefractions were pooled, stored at 4° C., and then assayed for totalNADP-MDH activation activity.

Preparation of Heat-Treated Extracts

Approximately 10 grams of barley grains were ground to powder for about30 sec in a coffee grinder and extracted by shaking for 1 hr at roomtemperature in 50 mL buffer as above. The slurry plus the rinse wassubjected to centrifugation at 27,000×g for 20 min and the supernatantsolution decanted through glass wool. A 20 mL aliquot of each sample washeated at 65° C. until sample temperature reached 60±1° C. (˜10 min).The sample was held at 60° C. for 10 additional min, followed by coolingin an ice/water bath. The cooled sample was centrifuged and thesupernatant solution was concentrated by sucrose as above and stored at−20° C. Frozen samples were thawed and clarified by centrifugation at14,000 rpm for 10 min at 4° C. Total TRXh activity was estimated on theconcentrated, supernatant fractions.

NADP-Malate Dehydrogenase Activation Assay

Thioredoxin h activity was assayed as previously described (Florencio etal., 1988; Johnson et al., 1987a). Fifty to 120 μl of extract (dependingon activity) was preincubated with DTT, and 0.16 to 0.32 μl of thepre-incubation mixture was used for the NADP-MDH assay. Control assayswere conducted on identical fractions in the absence of NADP-MDH.Western blot analysis was conducted as described above except that 10 to20 % SDS-polyacrylamide gels were used for electrophoresis and transferto nitrocellulose paper was for 4 hrs at 40 V.

Sequential Extraction of Multiple Protein Fractions

Ten grams of barley grain were sequentially extracted for albumin(H₂O-soluble), globulin (salt-soluble), hordeins (alcohol-soluble) andglutelins (Shewry et al., 1980). Barley powder was stirred with 0.5 MNaCl for 1 h at 25° C. to remove salt-soluble proteins. Two sequentialhordein fractions were extracted from the residue with 50% propanol inthe absence (hordein-I) and presence (hordein-II) of 2% (v/v)2-mercaptoethanol. Glutelins were extracted from the residue with 0.05 Mborate buffer, pH 10, containing 1% (v/v) 2-mercaptoethanol and 1% (v/v)sodium dodecylsulphate.

In Vitro Monobromobimane (mBBr) Labeling of Proteins

Immature, mature, or germinating seeds from nontransformed andtransgenic plants were ground in 100 mM Tris-HCl buffer, pH 7.9.Reactions were carried out following the protocol of Kobrehel et al.,(1992). Seventy microliters of the buffer mixture containing a knownamount of protein was either untreated or treated with DTT to a finalconcentration of 0.5 mM. After incubation for 20 min, 100 nmol of mBBrwas added, and the reaction was continued for another 15 min. To stopthe reaction and derivatize excess mBBr, 10 μl of 10% SDS and 100 μl of100 mM 2-mercaptoethanol were added. The samples were applied to a 15%SDS-PAGE gel. Fluorescence of mBBr was visualized by placing gels on alight box fitted with a UV light source (365 nm). Protein determinationwas carried out by the Bradford dye binding method (Bradford 1976) usingbovine serum albumin or gamma globulin as standards.

Assay of Pullulanase and its Inhibitor

To measure pullulanase activity, grain was germinated in a dark chamberand retained for up to 5 days at 25° C. as described (Kobrehel et al.,1992.; Lozano et al., 1996.). A set of plates from each line was removedfor extract preparation each day. Cell-free endosperm extracts wereprepared from lots of 10-20 germinated grains of equivalent root andcoleoptile length within a given cohort. Endosperm was separated fromthe embryo and other tissues and added to Tris-HCl buffer (50 mM, pH7.9) supplemented with 1 mM EDTA and 0.5 mM PMSF (1:3 to 1:6, wt/volratio of tissue to buffer depending on developmental stage). Aftergrinding in a mortar on ice, the sample was clarified by centrifugation(10 min at 24,000×g); the supernatant fraction was recovered and storedin 0.5-ml aliquots −80° C. for pullulanase spectrophotometric or gelassays.

Pullulanase activity was determined spectrophotometrically at 37° C. bymeasuring dye released after 30 min at 534 nm using red pullulan(Megazyme, Bray, Ireland) as substrate in 50 mM citrate-phosphate buffer(pH 5.2) (Serre et al., 1990). Pullulanase also was assayed on nativeactivity gels of 7.5% acrylamide, 1.5 mm thickness, containing 1% redpullulan (Furegon et al., 1994.). Gels were scanned using a Bio-Rad GelDoc 1000 and analyzed using Bio-Rad MULTI ANALYST, version 1.0.2.Pullulanase inhibitor activity was determined on fractions heated toinactivate pullulanase (70° C. for 15 min) by measuring their ability toinhibit added purified barley malt pullulanase. Endogenous pullulanaseactivity was shown to be completely eliminated by this heat-treatmentwhile the inhibitor activity was not affected (Macri et al., 1993;MacGregor et al., 1994).

Alpha-Amylase Activity in Barley Grain Overexpressing Thioredoxin h

Amylase activity from the null segregant and homozygous barley grainswas analyzed during germination and early seedling growth by using gelscontaining starch. Native polyacrylamide electrophoresis gels [6%acrylamide, 1.5 mm thick] were prepared and developed according to themethod of Laemmli (1970) except that SDS was omitted from all solutions.The separating gel contained 0.5% soluble starch (Lintner potato starch,Sigma Chemical Co., St. Louis, Mo.). Lyophilized samples were dissolvedin distilled H₂O and mixed 1:1 with a buffer consisting of 0.25 MTris-HCl, pH 6.8, 50% glycerol, 0.04% bromophenol blue, and 3 mM CaCl₂.Fifty micrograms of sample protein were loaded in each lane.Electrophoresis was carried out at 80 milliamps per gel at 4° C. untilthe dye front was at the edge of the gel (usually 4 to 5 hours). Afterelectrophoresis, the gels were incubated in 100 ml of 0.1 M succinatebuffer, pH 6.0, for 1-2 hours at 37° C. The gels were then stained for 5min in a solution containing 2.5 mM I₂ and 0.5 M KI. Gels were washed indistilled H₂O. Except for the white regions containing amylase activity,gels were stained dark blue.

Isoelectricfocusing (IEF)

For determination of alpha-amylase isozyme patterns, extracts from bothdry and germinating grain of transformed and control (untransformed)barley were separated by electrophoresis at 4° C. [1.0 mm thick, pH 3-10isoelectric focusing (IEF) polyacrylamide gels, using the X cell IIsystem (NOVEX, San Diego, Calif.)]. Cathode buffer contained 20 mMarginine, and 20 mM lysine; anode buffer was 7 mM phosphoric acid.Samples were mixed 1:1 and 2× IEF sample buffer pH 3-10 (NOVEX). Aftersample application (20 μg/lane) gels were developed at constant voltage[100 V for 1 hr, 200 V for an additional 1 hr, and 500 V for 30 min].IEF standards (Bio-Rad) were used to determine the pH gradient of thegels.

Multiple Antibody Probing of IEF Gels

Western blot analysis of alpha-amylase isozymes was performed using aMini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Seed extractsfrom the null segregant and homozygous lines overexpressing wheatthioredoxin h were separated by IEF gels as described above. Proteinswere transferred to nitrocellulose at a constant voltage of 100 V for 1hr at 4° C. using 0.75% acetic acid as blotting buffer. Nitrocellulosewas blocked with 5% powdered milk in Tris buffer solution (20 mMTris-HCl, pH 7.5, supplemented with 0.15 M NaCl) for 1 hr at roomtemperature, incubated with primary antibody for 4 hours at roomtemperature and then with secondary antibody for 1 hour at roomtemperature. Primary antibody was anti-barley alpha-amylase B diluted1:1000; secondary antibody was goat anti-rabbit alkaline phosphatase(Bio-Rad) diluted 1:3000. Blots were developed in NBT/BCIP alkalinephosphatase color reagent (according to Bio-Rad instructions) therebyrendering the cross-reacted alpha-amylase bluish-purple. To achieve fullidentity of isozyme pattern, blots were probed a second time withanother primary antibody, anti-alpha-amylase A (diluted 1:1000) and thesecondary antibody (as above). This time blots were developed inNaphthol Phosphate/Fast Red alkaline phosphatase color reagent(according to Bio-Rad instructions) which gave a pink stain to thealpha-amylase A. The blot shown was subject to this dual probingprocedure.

B. Results and Discussion

Production of Transgenic Plants

One day after bombardment, the whole embryos were transferred onto DCmedium with 5 mg/L bialaphos. At transfer to the second selection plate(5 mg/L bialaphos), all material from individual callusing embryos wasbroken into small pieces (2-4 mm) using forceps and maintainedseparately. During the subsequent two to five selection passages on 5mg/L bialaphos (at 10-20 d intervals). callus pieces showing evidence ofmore vigorous growth were transferred to new selection plates. Duringthe second round of selection, some pieces of callus were inhibited ingrowth and in some cases pieces turned brown. In general, transformedtissues were observed after three or more rounds of selection. Thebialaphos-resistant tissues were transferred onto an intermediatemedium, DBC2 or DBC3 (Cho et al., 1998a-c) with bialaphos (5 mg/L), andgrown for 1 to 2 months before regeneration on FHG medium containing 3mg/L bialaphos. Green plantlets were transferred into Magenta boxescontaining 3 mg/L bialaphos. Twenty-eight independent putativelytransformed, regenerable lines were produced after bialaphos selection(shown in Table 1). TABLE 1 Transgenic Barley Lines Transformed withWheat Thioredoxin h Gene. DNA PCR Plasmids for (T₀ leaf) TRXh ExpressionBombardment Transgenic Barley Line bar wtrxh in T₁ seeds Ploidy CommentspdBhWTRXN-1 + GPdBhBarWTRX-1 + + n.d. Tetraploid pAHC20GPdBhBarWTRX-2 + + + Tetraploid GPdBhBarWTRX-3 + + + DiploidGPdBhBarWTRX-5 + + + Tetraploid Sterile GPdBhBarWTRX-16 + − n.d.Tetraploid GPdBhBarWTRX-17 + + n.d. Tetraploid GPdBhBarWTRX-19 + + +Diploid GPdBhBarWTRX-20 + + + Diploid GPdBhBarWTRX-22 + + + DiploidGPdBhBarWTRX-23 + + + Diploid pdBhssWTRXN3-8 + GPdBhssBarWTRX-1 + − −Diploid pAHC20 GPdBhssBarWTRX-2 + + + Diploid HomozygousGPdBhssBarWTRX-3 + + − Diploid GPdBhssBarWTRX-7 + + + DiploidGPdBhssBarWTRX-9 + + n.d. Tetraploid GPdBhssBarWTRX-11 + + − DiploidGPdBhssBarWTRX-13 + + + Tetraploid GPdBhssBarWTRX-14 + + + DiploidGPdBhssBarWTRX-20 + + + Tetraploid GPdBhssBarWTRX-21 + + n.d. TetraploidSterile GPdBhssBarWTRX-22 + + + Tetraploid GPdBhssBarWTRX-29 + + +Diploid Homozygous pDhWTRXN-2 + GPDhBarWTRX-5 + + + Tetraploid pAHC20GPDhBarWTRX-7 + + + Diploid GPDhBarWrRX-8 + + + DiploidGPDBhBarWTRX-9 + + + Diploid Homozygous GPDBhBarWTRX-22 + + + DiploidSterile pGlbWTRXN-1 + GPGlbBarWTRX-1 + + + Diploid pAHC20*n.d.: not determined

Analysis of T₀ Plants and their Progeny

PCR analysis was performed using two sets of WTRXh primers and one setof BAR primers (see FIG. 1). PCR amplification resulted in 0.4-kb intactwtrxh or 0.14 kb truncated wtrxh and 0.34-kb internal bar fragments fromtransgenic lines. Of the 28 lines tested, 28 yielded bar fragments fromT₀ leaf tissue and 26 produced PCR-amplified fragments for wtrxh, givinga 93% co-transformation frequency. Nine lines were transformed withpdBhWTRXN-1, eleven with pdBhssWTRXN-8, five with pDhWTRXN-2 and onewith pG1bWTRXN-1 (see Table 1). Three lines (GPdBhBarWtrx-5,GPdBhssBarWtrx-21 and GPDhBarWtrx-22) were sterile. Seeds of T₁ plantsand their progeny from selected wtrxh-positive lines were planted inorder to screen for homozygous lines. Homozygous lines and nullsegregants were obtained from GPdBhssBarWtrx-2, -29 and GPDhBarWtrx-9(see Table 1).

Cytological Analysis of Transgenic Plants

Chromosomes were counted in root meristem cells of independentlytransformed T₀ barley plants. Out of 28 independent transgenic linesexamined. 17 lines had the normal diploid chromosome complement(2n=2x=14), while the remaining 11 lines were tetraploid (2n=4x=28) (seeTable 1).

Characterization and Content of WTRXh Produced in Transgenic Seed

As discussed above, several stably transformed barley lines wereobtained that express wheat thioredoxin h. As seen in FIG. 2, the stableintroduction of the wtrxh linked to the B1-hordein promoter with thesignal peptide sequence resulted in greatly enhanced expression ofactive WTRXh in transgenic barley seed.

Analysis by western blot of soluble protein fractions of the three linesin which the thioredoxin gene was linked to a signal sequence(GPdBhssBarWtrx-22, GPdBhssBarWtrx-29 and GPdBhssBarWtrx-7) showeddifferences in the level of expression (shown in Table 2). LineGPdBhssBarWtrx-22, GPdBhssBarWtrx-29 and GPdBhssBarWtrx-7, respectively,showed 22 times, 10 times and 5.5 times more WTRXh protein thannontransformed control seeds. The analyses showed that the thioredoxincontent of the null segregant (GPdBhssBarWtrx-29-11) was approximatelyhalf that of the corresponding control. The three lines generated fromthe construct in which the thioredoxin gene was not associated with asignal sequence were also compared to nontransformed control barley seedand they exhibited the following increases in TRXh levels as indicatedby the western blot analyses: GPDhBarWtrx-9: 12 times; GPDhBarWtrx-5:6.3 times; GPdBhBarWtrx-2: 6.4 times. When probed on Western Blots, thetransgenic lines show two bands while the control barley generally showsonly one and in some cases a second minor band. Furthermore, the tissuesfrom the transgenic lines were characterized by a band that did notcorrespond to either of the barley bands but did correspond to wheatthioredoxin h. These data indicate that the protein introduced bytransformation is wheat thioredoxin h. TABLE 2 Western Blot Analyses ofOverexpression of Wheat Thioredoxin h in Barley. % Fold Increase BarleyLine Volume Scanned (or Decrease) Non-Transformed Control: GoldenPromise 1.46 1.0 Transformed with Signal Sequence: GPdBhssBarWtrx-2232.44 22 GpdBhssBarWtrx-29 14.62 10 GpdBhssBarWtrx-7 7.99 5.5Transformed without Signal Sequence: GPDhBarWtrx-9 17.69 12GPDhBarWtrx-5 9.20 6.3 GPdBhBarWtrx-2 9.29 6.4 Null Segregant:GPdBhssBarWtrx-29-11-10 0.93 (0.64)

The Wheat Thioredoxin h in Barley Grains is Biologically Active

Because of interference from other enzymes that oxidize NADPH, theactivity of TRXh cannot be accurately assayed in crude extracts, therebynecessitating its partial purification. Partially purified extracts ofthe different transgenic and control lines were prepared from 15 gramsof seed using ammonium sulfate fractionation and gel filtrationchromatography. Activity was measured with an NADP-MDH activation assay.Profiles based on these assays show that the activity of TRXh in thetransformed seed is much higher than in the nontransformed control (seeFIG. 2). The activity results are summarized in Table 3.

Total WTRXh activity from the seeds of two lines transformed with theB1-hordein promoter and the signal sequence (GPBhssBarWtrx-3;GPdBhssBarWtrx-29) is about 4- to 10-fold higher, respectively, thanthat of control, nontransformed seed. Total activity from a linetransformed with the D-hordein promoter without the signal sequence(BGPDhBbarWtrx-5) is only slightly higher (1.25-fold) than that of thenontransformed control (see Table 3). In the transgenics, the specificactivity of thioredoxin is generally about 0.128 A_(340 nm)/min/mgprotein or about two fold over null segregants. TABLE 3 Summary of TotalBuffer-Extracted Protein and Total Thioredoxin Activity from ActiveFraction after Gel Filtration. Specific Total Total Activity, Activity,Barley Line Protein, mg A₃₄₀/min A₃₄₀/min/mg Control (GP 4-96)  102.6(1.00)*  7.4 (1.00)*  0.064 (1.00)* GPDhBarWtrx-5 171.2 (1.67) 9.2 (1.2)0.054 (0.8)  GpdBhssBarWtrx-29 149.1 (1.45) 72.0 (9.7)  0.483 (7.5) GpdBhssBarWtrx-3 231.3 (2.25) 27.7 (6.4)  0.794 (12.4)*Numbers in brackets are fold increase over that of the control.

The transformed barley grains analyzed so far appear to have more totalbuffer-extracted protein than control, nontransformed seed (Table 3).

The transformed grains have a thioredoxin content of at least about10-15 μg thioredoxin/mg soluble protein(about 2-8 μg thioredoxin/mgtissue) or about two-fold higher than the null segregant.

Because of the tediousness of the (NH₄)₂SO₄ procedure and therequirement for large quantities of seed, the original extractionprocedure was modified to include a heat treatment step. This change wasbased on the fact that E. coli WTRXh is stable after treatment at 60° C.for 10 min (Mark and Richardson, 1976). Results on WTRX from twodifferent transgenic barley seeds (GPdBhBarWtrx-3, GPdBhssBarWtr-29)showed no significant difference in activity between the heat treatedand non-heat treated extracts (FIG. 3). In addition heat-treatmentdecreased the endogenous, nonspecific activity in this assay, therebyincreasing the reliability of the measurements.

Ten different barley lines (transformed and nontransformed) wereextracted using the heat-treatment step and assayed with the NADP-MDHassay; the results are summarized in Table 4. In general, total WTRXhactivities in seeds from lines transformed with the B-hordein promoterand signal sequence linked to wtrxh are much higher (4- to 35-fold) thanin seeds from lines transformed with the same promoter without signalsequence linked to wtrxh or in seeds from the nontransformed control(Table 4). At this point it is not known whether all expressed wheatWTRXh in barley seeds is heat stable. TABLE 4 Relative Total ThioredoxinActivity in Different Transgenic Barley Lines. Total Total Specific LineDesignation Protein (%) Activity (%) Activity (%) Non-transgenic controlGP4-96 100 100 100 Bar Gene Only GPBar-1 92 120 131 Without SignalSequence GPdBhBarWtrx-1 101 192 190 GPdBhBarWtrx-22 113 151 133GPdBhBarWtrx-23 118 180 153 With Signal Sequence GPdBhssBarWtrx-2 1371650 1203 GPdBhssBarWtrx-14 122 1723 1418 GPdBhssBarWtrx-20 147 440 299GPdBhssBarWtrx-22 154 3470 2245 GPdBhssBarWtrx-29 108 1316 1219One hundred percent of (a) total protein, mg; (b) total activity,nmol/min; and (c) specific activity, nmol/min/mg protein of thenon-transgenic control are: (a) 116.4; (b) 157.38 (c) 1.52, respectively

Of the stably transformed lines that expressed wheat thioredoxin h, onaverage, its level was found to be higher in transformants that had thesignal peptide-containing constructs than to those that did not (Table4). Western blot analysis of soluble protein fractions from heterozygousmixtures of seeds from three of the lines, GPdBhssBarWtrx-7,GPdBhssBarWtrx-29, and GPdBhssBarWtrx-22 showed 5.5 times, 22 times, and10 times more thioredoxin h, respectively, than nontransformed controlgrain (Table 2). The thioredoxin content of the null segregant(GPdBhssBarWtrx-29-11-10) was about half that of the corresponding,nontransformed control.

Extracts from barley typically showed one immunologically reactive band(identified by B in FIG. 4A, lanes 1 and 6) but in some transfers showeda second faint, faster moving band (FIG. 4B, lane 2). Tissues fromtransgenic lines overexpressing wtrxh were characterized by a band thatdid not correspond to either of the two counterparts in barley, butrather to thioredoxin h from wheat. The difference between theoverexpressed 13.5-kDa wheat and the endogenous 13.1-kDa barleythioredoxin h is particularly pronounced in the barley line transformedwith the nontargeted thioredoxin h gene (FIG. 4A, line 5 and FIG. 4B,lane 1). Repeated analyses of the various transgenic lines by SDS/PAGEled to the conclusion that the band identified in FIGS. 4A-B by Wcorresponds to the bread wheat wtrxh introduced by barley. Independentbiochemical assays with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)(Florencio et al., 1988.) confirmed the ability of barley NTR to reducewheat thioredoxin h (data not shown).

Because of their value in assessing biochemical attributes of the grain,homozygous wtrxh lines were identified and analyzed by Western blot. Thetwo lines identified as homozygous showed both enhanced expression ofthioredoxin h relative to that of their heterozygous parents andnontransformed controls. Analysis of GPdBhssBarWtrx-29-3 is shown inFIG. 5. It is noted that demonstration of the thioredoxin h present inthe nontransgenic control and null segregant grains (not apparent in theexposure shown in FIG. 4) required conditions that led to overexposureof the enriched transgenic preparations. Thioredoxin in the parentheterozygous grain was shown to be biochemically active.

Pullulanase and Pullulanase Inhibitor Activity in Barley GrainOverexpressing Thioredoxin h

Pullulanase is an amylolytic enzyme present in cereal grain, which has adisulfide inhibitor protein (Macri et al., 1993.; MacGregor et al.,1994.), the activity of which is linked to thioredoxin (Wong et al.,1995.). Thioredoxin reduced by NADPH via NTR, reduces the disulfidebonds of the inhibitor, allowing the targeted pullulanase enzyme to beactive. Because of this relationship, it was of interest to determinethe activity of pullulanase in the thioredoxin h-overexpressingtransformants.

Spectrophotometric assays (FIG. 8A) of extracts from transformed grainof a homozygous line (GPdBhssBarWtrx-29-3) overexpressing thioredoxin hshowed a 3- to 4-fold increase in pullulanase activity on the fifth dayafter initiation of germination relative to its null segregant.Confirmatory results were obtained in a separate experiment with nativeactivity gels. The increase in activity was apparent either when gelswere viewed directly (FIG. 8B) or when the activity on the gels wasassessed by scanning and integrating the clarified bands (FIG. 8C). Ahomozygous line isolated from a different, independent transformationevent (GPdBssBarWtrx-2-1-15) showed a similar response (data not shown).The transgenic plants expressed an pullulanase activity of about 1-2Absorbance units at 534 nm/30 min/mg protein, which is about two-foldhigher than null segregants.

Pullulanase inhibitor activity was determined on fractions heated toinactivate pullulanase (70° C. for 15 min) by measuring the inhibitionof the fractions on added purified barley malt pullulanase. Theendogenous pullulanase activity was shown to be completely eliminated bythis heat treatment whereas inhibitor activity was not affected (Macriet al., supra; MacGregor et al., supra). Analysis of comparable grainextracts revealed that the pullulanase inhibitor was inactive on thefourth and fifth days after water addition in both the transformant andnull segregants. These results thus demonstrate that the increase inpullulanase activity observed after the third day is not caused byenhanced inactivation of the inhibitor in the transgenic grain. It ispossible that thioredoxin acts either by increasing the de novosynthesis of pullulanase (Hardie et al., 1975.) or by lowering thebinding of the mature enzyme to the starchy endosperm. There is evidencethat some of the pullulanase of the mature endosperm is present in boundform and can be solubilized by reducing conditions (Sissons et al.,1993.; Sissons et al., 1994.).

Alpha-Amylase Activity in Barley Grain Overexpressing Thioredoxin h

Alpha-amylase, also an amylolytic enzyme that is induced by gibberellicacid like pullulanase, has long been considered key to germination. Thesynthesis of the major (B) and minor (A) forms of this enzyme are knownto be triggered by the hormone, gibberellic acid (GA). In addition,alpha-amylase activity is increased in vitro by the reductiveinactivation of its disulfide inhibitor protein by thioredoxin h (in thepresence of NADPH and NADP-thioredoxin reductase). The present resultswith transformed barley seeds show that, like pullulanase, thioredoxin hexpression alters alpha-amylase activity. In this case, the appearanceof the enzyme during germination is accelerated and its abundance andactivity are increased.

FIG. 9A-D shows the early increase in both the abundance and activity ofalpha-amylase (A+B forms) during gemination and seedling development.Based on the antibody response in western blots, alpha-amylase was firstdetected 3 days after the onset of germination in the transgenic grainFIG. 9C) whereas the enzyme did not appear until the fourth day in thenull segregant (FIG. 9A). The onset of activity (based on the activitygel) followed a similar pattern (FIG. 9B and FIG. 9D). The mobility ofthe enzyme in the activity gel also reflected the early induction ofactivity in the transgenic grain (FIG. 10). That much of this increasein activity seen early on was due to the B (a gibberellic acid-linkedform) is supported by FIG. 11. Here, one can also see that the level ofthe minor A form of the enzyme (also gibberellic acid dependent) wasincreased in grain overexpressing thioredoxin h. Again, the appearanceof significant levels of the major (B form) alpha-amylase enzyme wasadvanced by 1 day.

Germination of Barley Grains Overexpressing Thioredoxin h

All operations were carried out at 25° C. (unless otherwise specifiedbelow) under conditions described by Kobrehel et al., 1992 and Lozano etal. 1996. Grains were surface sterilized by continuous stirring in 0.25%bleach for 30 min: Bleach was removed by extensive washing withsterilized distilled water. Thirty sterilized null segregant(GPdBhssBarWtrx-29-22-10, in which the transgene was removed by crossingwith a self-polinated plant from the same line) and thirty sterilizedhomozygous (GPdBhssBarWtrx-29-3) seeds were placed in each of a seriesof plastic Petri dishes (12.5 cm diameter) fitted with three layers ofWhatman #1 filter paper moistened with 15 ml sterile distilled water.Plates were wrapped with aluminum foil and grain was germinated in adark chamber at 20° C. for up to 7 days. One plate was read at each timepoint shown in FIG. 21. Percent germination, in the first day (from thestart of incubation up to 24 hours), was determined by observing theemergence of the radicle. On the subsequent days, percent germinationrepresents seedling growth as determined by measuring the length ofcoleoptile and roots of the germinated grains.

The results, shown in FIG. 21, indicate that germination in transgenicbarley overexpressing wheat thioredoxin h is detected about 16 hoursafter the onset of incubation in about 25-30% of the seeds. In contrast,no germination in the null segregant was detected at 16 hours but isfirst detected 8 hours later, on Day 1. Therefore, in the transgenicgermination is advanced about 8 hours. However, on Day 1 germination wasdetected in approximately 70% or about twice the number of transgenicgrains in comparison to their null segregant counterparts. It isinteresting to note that the onset of germination in the transgenicsparallels the onset of the detection of alpha amylase as shown in FIG.10.

Sequential Extraction of Grain Proteins from Transgenic Barley Grains.

Isolated endosperm from 10 dry grains or seedlings (germinated asdescribed above) were ground with mortar and pestle 4° C. with 3 mlTris-HCl buffer as indicated below. The separate mixtures of homozygousGPdBhssBarWtrx-29-3 and null segregant GPdBhssBarWtrx-29-22-10 grainswere placed in a 5-ml screw-top centrifuge tube. Grains weremechanically shaken for 30 minutes and then centrifuged for 10 min at24,000×g. The supernatant fraction (buffer-soluble) was decanted andsaved for analysis and the residue was extracted sequentially with thefollowing solvents for the indicated times: [1] 0.5 M NaCl (30 min); [2]water (30 min); [3] 2×50% propanol (2 hr); [4] 2×50% propanol+2%2-mercaptoethanol (MET) (2 hr); and [5] 0.5 M borate buffer, pH 10,containing 1% SDS and 2% 2-mercaptoethanol (2 hr). Supernatant fractionsof all extracts were determined for volume and protein content (byCoomassie dye binding method), then were stored at −20° C. until use. Byconvention, the fractions are designated: [1] albumin/globulin(buffer/salt/water); [2] Hordein I (propanol); [3] Hordein II(propanol+MET); and [4] glutelin (Borate/SDS/MET) (Shewry et al., 1980).These fractions were used to determine, protein content, thedistribution of proteins between the water soluble and insolublefractions, the total extractable protein, and reduction with NADPH.

To determine the in vivo redox status of protein from transgenic barleygrain during germination and seedling development, the extractionprocedure was repeated except that 2 mM mBBr was included in the Trisgrinding buffer and the grinding was under liquid nitrogen. The mBBrderivatized proteins were electrophoresed on SDS-polyacrylamide gels(1.5 mm thickness, 10-20% gels, pH 8.5 (Laemmli, 1970). Gels weredeveloped for 16 hr at a constant current of 8 mA. Followingelectrophoresis, gels were placed in 12% (w/v) trichloroacetic acid andsoaked for 4 to 6 hr with one change of solution to fix the proteins;gels were then transferred to a solution of 40% methanol/10% acetic acidfor 8 to 10 hr with agitation to remove residual mBBr. The fluorescenceof mBBr (both free and protein bound mBBr), was visualized by placinggels on a light box fitted with an ultraviolet light source (365 nm).Following removal of the excess (free) mBBr, images of gels werecaptured by Gel Doc 1000 (Bio-Rad).

To ascertain the equivalent protein amount of loaded extracts, SDS-gelswere stained with Coomassie Brilliant Blue G-250 in 10% acetic acid for30 min, and destained in 10% acetic acid for 30 min with the aid of amicrowave oven. Protein stained gels were captured by Gel Doc 1000 asabove.

The quantification of fluorescence (pixel×mm×mm) and protein (opticaldensity×mm×mm) on gels were carried out by a software program for imageanalysis—Multi-Analyst, version 1.0 (Bio-Rad). Relative reduction wasexpressed as the ratio of fluorescence to protein.

The results of two experiments shown in Table 5, Table 6, and Table 7demonstrate an increase in the total protein on a percent grain and apercent weight basis in the transgenic barley as compared to the nullsegregant. The transgenic have a thioredoxin content that is at leasttwo-fold higher (10-15 μg/mg soluble protein; 2-8 μg/gram tissue) thanthe null segregant. The data indicate that this increase in totalextractable protein is the result in redistribution of the protein tothe most soluble albumin/globulin fraction. The redistribution of theprotein to the soluble fraction increase in the transgenics is at least5% higher than the controls. TABLE 5 Protein Content of VariousFractions in Transgenic Barley Grain Overexpressing Wheat Thioredoxin hExperiment I* Null Segregant Homozygous Protein Fraction mg/seed mg/grammg/seed mg/gram Albumin/Globulin 0.462 12.25 0.546 13.58 Hordein I 0.2396.34 0.322 8.01 Hordein II 0.136 3.61 0.094 2.34 Glutelin 0.110 2.920.097 2.41 Total Extractable Protein 0.947 25.12 1.059 26.34*Weight per 10 seeds is 0.377 and 0.402 full null segregant andhomozygous line of transgenic barley

TABLE 6 Protein Content of Various Fractions in Transgenic Barley GrainOverexpressing Wheat Thioredoxin h Experiment II** Null SegregantHomozygous Protein Fraction mg/seed mg/gram mg/seed mg/gramAlbumin/Globulin 0.691 20.03 1.044 27.12 Hordein I 0.373 10.81 0.36810.03 Hordein II 0.254 7.36 0.240 6.23 Glutelin 0.066 1.91 0.062 1.61Total Extractable Protein 1.384 40.11 1.732 44.99*Weight per 10 seeds is 0.377 and 0.402 for null segregant andhomozygous line of transgenic barley

TABLE 7 Percent Increase of Extractable Protein in Homozygous Line%/(grain basis %/mass basis Experiment I 12 4.9 Experiment II 25 12

Analysis of the relative redox status (SH:SS) of protein fractions intransgenic and null segregant barley grains during germination and asdry grains are shown in FIG. 22. In dry transgenic grain, the greatestincrease in reduction relative to the null segregant was observed in thehordein I fraction. This increase was paralleled by decreases in therelative redox status in the hordein II and glutelin fractions while therelative redox status of the albumin/globulin fraction was unchanged.The relative redox status of the transgenic in comparison to the nullsegregant is at least 5:1.

During germination, the albumin/globulin fraction progressivelyincreases, reaching a relative redox ratio of about 1.5 on Day 4. Therelative redox status of the hordein II and glutelin fractions alsoincreased during germination but only reached parity with the nullsegregant. In contrast the relative redox status of the hordein Ifraction was highly variable.

According to the above example, other types of plants, are transformedin a similar manner to produce transgenic plants overexpressingthioredoxin, such as transgenic wheat, described below, rice, maize,oat, rye sorghum (described below), millet, triticale, forage grass,turf grass, soybeans, lima beans, tomato, potato, soybean, cotton,tobacco etc. Further, it is understood that thioredoxins other thanwheat thioredoxin or thioredoxin h can be used in the context of theinvention. Such examples include spinach h; chloroplast thioredoxin mand f, bacterial thioredoxins (e.g., E. coli) yeast, and animal and thelike.

Example 2 Transgenic Wheat Grain Overexpressing Thioredoxin h andArabidopsis NTR

A. Materials and Methods

Plant Materials

Spring cultivar of wheat, Bobwhite, Anza and Yecora Rojo, were grown inthe greenhouse as described previously (Wan and Lemaux 1994; Lemaux etal. 1996). Ten- to 14-day-old germinating plants of a winter-wheatcultivar, Karl, were incubated at 4° C. for 45 to 60 days in the darkfor vernalization treatment.

Wheat Expression Vectors

For wheat transformation, synthetic green fluorescent protein gene[sfgp(S65T)], wheat thioredoxin h (wtrxh) or Arabidopsis ntr expressionvectors driven by barley endosperm-specific B₁- or D-hordein wereconstructed as follows:

-   -   (1) pDhSSsGFPN3-4: the chimeric DNA construct containing the        D-hordein promoter-signal sequence-sgfp(S65T)-nos was obtained        using a modified method of site-directed mutagenesis by PCR (Cho        and Lemaux 1997). The three-primer strategy was used. A shorter        fragment of 0.5-kb DHORSS was produced by PCR in the first        reaction using primers, Dhor4        (5′-agaaagcttggtaccCTTCGAGTGCCCGCCGAT-3′; SEQ ID NO:9) and        DhorSSsGFP1 R        (5′-GAACAGCTCCTCGCCCTTGCTCACAGCGGTGGTGAGAGCCACGAGGGC-3′; SEQ ID        NO:10), with the template pHor3-1 containing a genomic clone of        D hordein (Sørensen et al., 1996), and this first PCR product        (megaprimer) was diluted 50 times. DhorSSsGFP1R is an        overlapping primer which contain the sgfp(S65T) coding sequence        and a partial signal peptide sequence (underlined) from the        D-hordein promoter. For the second PCR reaction, five μl of the        diluted megaprimer (DHORSS), twenty ng of template        (pAct1IsGFP-1; Cho et al., 2000) and 40 pmol of external primers        [Dhor4 and Nos1R (5′-cggaattcGATCTAGTAACATAGATGACA-3′: SEQ ID        NO:17)] were mixed to a final volume of 100 μl in 1× PCR buffer;        pAct1IsGFP-1 contains synthetic gfp gene [sgfp(S65T)] (Chiu et        al., 1996) controlled by the rice actin1 promoter and its intron        and terminated by nos. The resulting chimeric PCR product was        digested with HindII and EcoRI and ligated into the        HindII/EcoRI-digested pBluescript II KS(+) vector, further        confirmed by DNA sequencing of the PCR-amplified fragment        [D-hordein promoter with its signal peptide sequence plus the        junction region with the 5′ sgfp(S65T)], and used for stable        transformation of wheat.    -   (2) pDhWTRXhN-2: the 384-bp wtrxh coding region was amplified by        PCR utilizing the plasmid pTaM13.38 (Gautier et al., 1998)        containing cDNA clone of wtrxh gene as a template to create XbaI        and SacI sites with primers Wtrxh1        (5′-atatctagaATGGCGGCGTCGGCGGCGA-3′; SEQ ID NO:5) and Wtrxh2R        (5′-atagagctcTTACTGGGCCGCGTGTAG-3′; SEQ ID NO:6), respectively        (FIG. 12); small letters contain a restriction enzyme site for        subcloning of the DNA construct containing the wtrxh gene and        underlined letters indicate the wtrxh sequences. The ATG        initiation codon for wtrxh expression was included in the Wtrxh1        primer. PCR reactions were performed on a thermocycler (MJ        Research Inc., Watertown, Mass.) using recombinant Taq DNA        polymerase (Promega, Madison, Wis.) in a 100-μl reaction volume.        The reaction buffer contained 10 mM Tris-HCl (pH 9.0), 50 mM        KCl, 1.5 mM MgCl₂, 0.1% Triton-X-100, and 50 μM of each        deoxyribonucleoside triphosphate. PCR conditions were 25 cycles        of 94° C. for 1 min, 55° C. for 1 min and 72° C. for 2 min, with        a final extension step at 72° C. for 7 min. The wtrxh fragment        amplified with primers Wtrxh1 and Wtrxh2R was purified from a        0.7% agarose gel using QIAquick® gel extraction kit (Qiagen        Inc., Chatsworth, Calif.), digested with XbaI and SacI and        ligated into XbaI/SacI digested pUC19 to generate the pWTRXh-1        plasmid. Nucleotide sequences of the PCR-amplified wtrxh coding        region were determined by dideoxynucleotide chain termination        method using Sequenase according to manufacturer's instructions        (United States Biochemical, Cleveland, Ohio) with        double-stranded plasmid templates and regularly spaced primers.        pDhWTRXN-2 was made by replacing the uidA gene in pDhGN-2        (containing barley endosperm-specific D-hordein promoter and nos        3′ terminator; M.-J. Cho, unpublished) with the XbaI/SacI        fragment containing wtrxh coding sequence from the pWTRXh 1.    -   (3) pdBhssWTRXhN3-8: primers Bhor7        (5′-GTAAAGCTTTAACAACCCACACATTG-3′; SEQ ID NO:7) and BhorWtrxh1R        (5′-CCGACGCCGCTGCAATCGTACTTGTTGCCGCAAT-3′; SEQ ID NO:8)        containing HindIII and Acyl sites, respectively, were used for        amplification of 0.49-kb B₁-hordein 5′ region including the        B₁-hordein signal peptide sequence using the λ2-4/HindIII        plasmid containing genomic clone of B₁-hordein (Brands et al.,        1985; Cho et al., 1997) as a template. The primer BhorWtrxhIR is        an overlapping primer containing the wtrxh coding sequence        (underlined) and a partial signal peptide sequence from the        B₁-hordein promoter without the ATG initiation codon for wtrxh.        pdBhssWTRXhN3-8 was made by replacing the D-hordein promoter in        pDhWTRXN-2 with the 0.49-kb PCR-amplified HindIII/Acyl fragment        containing B₁-hordein promoter with its signal peptide sequence        plus the junction region with the 5′ wtrxh. Thus, construct        pdBhWTRXN3-8 contains the barley endosperm-specific B₁-hordein        promoter with its signal peptide sequence, wtrxh and nos (FIG.        12). The signal peptide sequence containing the ATG initiation        codon was directly combined with the sequence of the wtrxh gene        (Gautier et al., 1998), without having extra amino acid        sequences between the two, in order to make WTRXh, protein        provide a precise cleavage site in the lumen of endoplasmic        reticulum (ER). The PCR-amplified fragment of the chimeric        product was confirmed by DNA sequencing.    -   (4) pKBhssWTRXN-2: pBhor-1 was digested with Sphl and SacI in        order to obtain the 0.55-kb 5′-flanking region of B₁-barley        hordein promoter. The 0.55-kb Sphl/SacI fragment was ligated        into pSPORT 1 (GIBCO BRL, Gaithersburg, Md.) to make pSPBhor-4.        pdBhssWTRN3-8 was digested with HindIII/EcoRI and the        HindIII/EcoRI fragment containing the 0.43-kb barley        endosperm-specific B₁-hordein promoter plus its signal peptide        sequence, wrxh and nos was ligated into the        HindIII/EcoRI-digested pSPBhor-4 to generate the pSPBhssWTRXN-4        plasmid. In order to remove ampicillin resistance gene, the        1.3-kb Sphl/EcoRI fragment of pSPBhssWTRXN-4 was ligated into        Sphl/EcoRI-digested pJKKmf(−) containing kanamycin resistance        gene to form pKBhssWTRXN-2. Thus, the kanamycin^(r)-backbone        construct, pKBhssWTRXN-2, contains the 0.55-kb 5′-flanking        region of the B₁-barley hordein promoter plus its signal peptide        sequence, wrxh and nos (FIG. 12).    -   (5) pDhAtNTR-4: pDhAtNTR-4 was made by replacing the wtrxh gene        in pDhWTRXN-2 (described above) with the PCR-amplified XbaI/SacI        fragment containing Arabidopsis ntr coding sequence from pAtNTR        (a gift from Dr. S. Y. Lee). Primers, AtNTR1        (5′-ggtctagaATGGAAACTCACAAAACC-3′; SEQ ID NO:18) and AtNTR2R        (5′-gggagctcTCAATCACTCTTACCCTC-3′; SEQ ID NO:20), were used for        amplification of the 1.009-Kb XbaI/SacI fragment containing        0.993-Kb Arabidopsis ntr coding sequence; small letters contain        a restriction enzyme site for subcloning of the DNA construct        containing Arabidopsis ntr gene and underlined letters indicate        the Arabidopsis ntr sequences. The Arabidopsis ntr fragment was        purified from a 0.7% agarose gel using QIAquick® gel extraction        kit, digested with XbaI and SacI and ligated into        XbaI/SacI-digested pDhWTRXN-2 to generate the pDhAtNTR-4        plasmid. Nucleotide sequences of the PCR-amplified Arabidopsis        ntr coding region were determined by DNA sequencing.

Stable Wheat Transformation

Stable transgenic lines of wheat transformed with pDhSSsGFPN3-4,pdBhssVVTRXhN3-8, pKBhssWTRXN-2 or pDhAtNTR4 were obtained using highlyregenerative, green tissues as transformation targets. Highlyregenerative tissues have a high percentage of totipotent cells capableof sustained cell division and competent for regeneration over longperiod. In order to induce highly regenerative green tissues, wholeimmature embryos (IEs; 1.0-2.5 mm) were aseptically removed, placedscutellum side down on DBC3 medium (callus-induction medium containing1.0 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L BAP and 5.0 μM CuSO₄;Cho et al., 1998a-c). Five to 7 days after initiation, germinatingshoots and roots were removed by manual excision. After 3 weeks ofincubation at 24±1° C. under dim light conditions (approximately 10 to30 μE, 16 h-light), highest quality tissues from the scutellum wasselected and maintained on DBC3 medium. Alternatively, highlyregenerative, green tissues were obtained from daughter tissues,oval-shaped tissues with highly embryogenic structures which wereemerged at the base of germinating shoots or from the outside layer ofthe tissues near the base of germinating shoots. Seven to 14 days afterinitiation, daughter tissues (2-4 mm in length) were isolated fromgerminating IEs by manual excision and transferred to fresh DBC3 medium.After an additional 3- to 4-week incubation, the tissues were selectedagain, broken into 2 to 4 pieces of about 3 to 5 mm in size andtransferred onto fresh medium. The tissues were maintained on freshmedium, subculturing at 3- to 4-week intervals.

Only good quality tissues were selected for bombardment. The highlyregenerative tissues (preferably about 3 to 4 mm in size) weretransferred for osmotic pretreatment to DBC3 medium containing equimolaramounts of mannitol and sorbitol to give a final concentration of 0.4 M.Four hours after treatment with the osmoticum, the tissues werebombarded as previously described (Wan and Lemaux 1994; Lemaux et al.1996). Gold particles (1.0 μm) were coated with 25 μg of a 1:1 or 1:2molar ratio of a mixture of pAct1lHPT-4 (or pUbilNPTII-1) and and one of4 plasmids, pDhSSsGFPN3-4, pdBhssWTRXhN3-8, pKBhssWTRXN-2 or pDhAtNTR-4,followed by bombardment using a PDS-1000 He biolistic device (BioRad,Inc., Hercules, Calif.) at 600 or 900 psi. The plasmid pAct1 lHPT-4contains the hygromycin phosphotransferase (hpt) coding sequence undercontrol of the rice actin1 promoter (Act1), its intron and the nos 3′terminator (Cho et al., 1998a-c). pUbilNPTII-1 contains the neomycinphosphotransferase (nptl) gene under control of the maize ubiquitinpromoter and first intron and terminated by nos. Sixteen to 18 hr afterbombardment, the bombarded tissues were placed to DBC3 medium withoutosmoticum and grown at 24±1° C. under dim light.

Following the initial 10- to 14-day culturing period, each regenerativetissue was broken into 1 to 3 pieces depending on tissue size andtransferred to DBC3 medium supplemented with 20-25 mg/L hygromycin B(Boehringer Mannheim, Mannheim, Germany) for selection for hpt or 30mg/L G418 (Sigma, Saint Louis, Mo.) for nptII. Three weeks after thefirst round of selection, the cultures were transferred to fresh DBC3medium containing 30 mg/L hygromycin B or 40 mg/L G418. From the thirdround selection, the tissues were subcultured and maintained on DBC3medium containing 30 mg/L hygromycin B or 40 mg/L G418 at 3- to 4-weekintervals. After the fourth or fifth round of selection, survivingtissues were transferred to DBC3 medium without selective agent.Following the identification of green tissues with sufficientregenerative structures on DBC3, the tissues were plated on solidregeneration medium without selective agent and exposed to higherintensity light (approximately 45-55 μE). After four weeks onregeneration medium (callus-induction medium without phytohormones), theregenerated shoots were transferred to Magenta boxes containing the samemedium without selective agent. When the shoots reached the top of thebox plantlets were transferred to the soil.

Polymerase Chain Reaction (PCR) and DNA Hybridization

Total genomic DNA from leaf tissues was purified as described(Dellaporta, 1993). To test tor the presence of wtrxh in genomic DNA ofputatively transformed lines, 500 ng of genomic DNA was amplified by PCRusing either of two primer sets, Wtrxh1(5′-ATATCTAGAATGGCGGCGTCGGCGGCGA-3′; SEQ ID NO:5) and Wtrxh2R(5′-ATAGAGCTCTTACTGGGCCGCGTGTAG-3′; SEQ ID NO:6) or Wtrxh4(5′-CCAAGAAGTTCCCAGCTGC-3′; SEQ ID NO:11) and Wtrxh5R(5′-ATAGCTGCGACAACCCTGTCCTT-3′; SEQ ID NO:19). The presence of hpt andnptII was tested by using each of the primer sets, HPT6F(5′-AAGCCTGAACTCACCGCGACG-3′; SEQ ID NO:21) plus HPT5R(5′-AAGACCAATGCGGAGCATATAC-3′; SEQ ID NO:22) (Cho etal., 1998a-c) andNPT1F (5′-CAAGATGGATTGCACGCAGGTTCT-3′; SEQ ID NO:15) plus NPT2R(5′-ATAGAAGGCGATGCGCTGCGAAT-3′; SEQ ID NO:16). Amplifications wereperformed with Taq DNA polymerase (Promega, Madison, Wis.) in a 25-μlreaction (Cho et al., 1998a-c). Twenty-five μl of the PCR product withloading dye was electrophoresed on a 1.0% agarose gel with ethidiumbromide and photographed using exposure to UV light. Presence of 0.4-and 0.14 kb fragments was consistent with an intact and truncated wtrxhfragments, repectively; 0.81-kb hpt and 0.76-kb nptII fragments for thepAct1lHPT-4 and pUbilNPTII-1 plasmids, were produced with hpt and nptIIprimers, respectively. Homozygous lines for wtrxh were screened usingT₁, T₂ or T₃ plants by PCR anlaysis.

GFP Expression Detection by Fluorescence Microscopy

GPF expression was monitored at higher magnification using a NikonMicrophot-5A fluorescent microscope equipped with a Nikon B-2A filterblock containing a 450-490 excitation filter and a BAS20 emissionbarrier filter (Cho et al., 2000).

Western Blot Analysis

Western blot analysis was performed on seeds from selected transgenicwheat lines as well as from control counterparts grown under the sameconditions. Thioredoxin h purified from seeds of a bread wheat cultivar,cv. Capitole, was used as a reference. Whole seeds were ground to a finepowder with a mortar and pestle under liquid nitrogen. Ten seeds wereused for each sample; the volume of extraction buffer [50 mM Tris HCl orphosphate buffer, pH 7.8, 0.5 mM phenylmethyl sulfonyl fluoride (PMSF),1 mM EDTA] varied from 2 to 4 ml depending on the number of seeds usedand the viscosity of the extract. Grinding was continued for anadditional min after buffer addition, the preparation was centrifuged at14,000×g for 10 min and the supernatant solution was saved as thesoluble (albumin-globulin) fraction. SDS-PAGE of the soluble fractionwas performed in 12-17% polyacrylamide gradient gels at pH 8.5 (Laemmli,1970). Equal amounts of protein (40 μg) of each sample quantitatedaccording to Bradford (1976) were diluted 1:2 v/v in Laemmli samplebuffer, boiled for 3 minutes, loaded onto gels and subjected toelectrophoresis at a constant current of 15 mA. Proteins weretransferred to nitrocellulose at a constant voltage of 40 V for 4 hoursat 4° C. using a Hoefer Transphor Transfer Unit (Alameda, Calif.) (allat 25° C.). Nitrocellulose was blocked with 5% powdered milk in TBS for2 hours, incubated in primary antibody for 4 hours and in secondaryantibody for 1 hour. The primary antibody was wheat anti-thioredoxin hII (Johnson et al., 1987b) diluted 1 to 500; secondary antibody was goatanti-rabbit alkaline phosphatase (Bio-Rad, Hercules, Calif.) diluted1:3000. Blots were developed in NBT/BCIP alkaline phosphatase colorreagent (Bio-Rad, Hercules, Calif.). Images were scanned using a Bio-RadGelDoc 1000 (Hercules, Calif.) and analyzed using Bio-Rad Multi Analyst,version 1.0.2.

B. Results and Discussion

Construction of Expression Vectors

To overexpress sGFP(S65T), WTRXh and AtNTR in wheat seed, fiveexpression constructs containing wtrxh driven by endosperm-specifichordein promoters, pDhSSsGFPN3-4, pDhWTRXN-2, pdBhssWTRXhN3-8,pKBhssWTRXN-2 or pDhAtNTR-4, were made. Out of five constructs, four(pDhSSsGFPN3-4, pdBhssWTRXhN3-8, pKBhssWTRXN-2 or pDhAtNTR-4; FIG. 12)were used for stable transformation of wheat.

Production of Transgenic Plants

Highly regenerative tissues (at least 1 tissue, preferably 50, and mostpreferably 500 of 3-4 mm in length) were bombarded and cultured on DBC3medium for the first 10 to 14 days in the absence of selection. For thesecond transfer (1st round selection), selection was on DBC3 mediumsupplemented with 25-30 mg/L hygromycin B for hpt selection or 30 mg/LG418 for nptII selection. At the second round selection, DBC3 mediumwith 30 mg/L hygromycin B or 40 mg/L G418 was used. From the 4thtransfer (3rd round selection) onward, the selection pressure wasmaintained at the same level. In general, hygromycin- or G418-resistanttissues with some green sectors were observed at the third roundselection. Putative transgenic calli with green sectors were maintainedand proliferated on the same medium without selective agent from afterthe fourth or fifth round of selection, until the green sectors formedfully developed regenerative structures. Green regenerative tissues wereregenerated on regeneration medium and the plantlets transferred to soilapproximately 3 to 4 weeks after growth on the same medium of theMagenta boxes. To date using this transformation protocol, we obtainedtwo independent Bobwhite lines, four transgenic Anza lines, twotransgenic Yecora Rojo lines transformed with pdBhssWTRXhN3-8, oneBobwhite line transformed with pKBhssWTRXN-2 and one Yecora Rojo linetransformed with pDhAtNTR-4 (Table 8). We also obtained two independentBobwhite lines transformed with pDhSSsGFPN3-4 (data not shown).

Endosperm-Specific Expression of Barley Hordein Promoter in TransgenicWheat

Expression of GFP driven by barley D-hordein promoter was foundspecifically in the endosperm tissue of developing wheat grains; GFPexpression was not observed in immature embryo tissues (FIG. 13)

Analysis of T₀ Plants and their Progeny

PCR analysis was performed using two sets of WTRXh primers and one setof AtNTR primers. PCR amplification resulted in 0.4-kb intact wtrxh or0.14-kb truncated wtrxh (FIG. 14) and 0.5-kb internal Atntr fragmentsfrom transgenic lines. Seeds of T₁ and their progeny from somewtrxh-positive lines were planted in order to screen homozygous lines.Homozygous lines and null segregants were obtained from AZHptWTR-1,AZHptWTR-21 and YRHptWTR-1 (Table 8). Other lines are currently beingscreened for homozygous lines.

Characterization of Wheat Thioredoxin h Produced in Transgenic Grain

Of the stably transformed lines that expressed wheat thioredoxin h, onaverage, its level was found to be higher in transformants. Western blotanalysis of soluble protein fractions from heterozygous mixtures ofseeds from three of these lines, AZHptWTR-1, AZHptWTR-21 and YRHptWTR-1,showed approximately 5 times, 20 times, and 30 times more thioredoxin h,respectively, than nontransformed control grain (FIG. 15A). Thethioredoxin content of the null segregant (YRHptWTR-1-2-1 to -3) wassimilar to that of the corresponding, nontransformed control (FIG. 15Aand B). TABLE 8 Summary of Transformation Experiments for Three WheatCultivars: Bobwhite, Anza and Yecora Rojo DNA PCR WTRXh or NTRCultivars/Plasmids for Transgenic (T₀ leaf) expression in T₁ bombardmentwheat lines hpt wtrx ntr seeds Comments BW/pAct1IHPT-4 + BWHptWTR-1 + +n.d. pdBhssWTRXhN3-8 BWHptWTR-3 + − n.d. BWHptWTR-4 + + n.d.BWHptWTR-5 + − n.d. AZ/pACT1IHPT-4 + AZHptWTR-1 + + + homozygouspdBhssWTRXhN3-8 AZHptWTR-11 + + + AZHptWTR-13 + + n.d. AZHptWTR-21 + + +homozygous YR/pACT1IHPT-4 + YRHptWTR-1 + + + homozygous pdBhssWTRXhN3-8YRHptWTR-2 + − n.d. YRHptWTR-8 + + n.d. BW/pUbiINPTII-1 +BWNptBhWTR- + + n.d. pKBhssWTRN-2 10 YR/pAct1IHpt-4 + YRHptAtNTR-1 + +n.d. pDHAtNTR-4BW, AZ and YR represent Bobwhite, Anza, Yocora Rojo, respectivelyn.d.: not determined

Example 3 Effect of Thioredoxin Reduction on Digestion of WheatGlutenins by Trypsin and Pancreatin

Sequential Extraction of Grain Proteins from Transgenic Wheat Grains

Transgenic grain (YRHptWTR-1-1) and null segregant (YRHptWTR-1-2) grainwere ground with a coffee grinder at room temperature. Ground powderfrom 10 grams of each line was placed in a 250-ml screw-top centrifugebottle and 60 ml of each extraction solution indicated below was added.The mixture was shaken mechanically and then centrifuged for 30 min at5,000×g. The supernatant fraction was decanted and saved for analysis,and the residue was mixed with the next solution. The powdered grain wasextracted sequentially with the following solvents for the indicatedtimes: [1] 2×0.5 M NaC1 (30 min); [2] 2×70% ethanol (2 hr); [3] 2×0.1 Macetic acid (2 hr). Supernatant fractions of all extracts were analyzedfor protein by the Coomassie dye binding method (Bradford, 1976) andthen were stored at −20° C. until use. By convention, the fractions aredesignated: [1] albumin/globulin (water/salt-water); [2] gliadin(ethanol); and [3] glutenin (acetic acid) (Kruger et al., 1988; Shewryet al., 1986). These fractions were used for digestion and skin tests inExample 5, below.

Digestion of Glutenins

For reduction of glutenins extracted as above from non-transgenic greenhouse plants, 4.2 μg NTR, 2.4 μg thioredoxin (both from E. coli), and 1mM NADPH were added to 240 μg of target protein and incubated in a 37°C. water bath for 45 minutes. NTS (NTR/thioredoxin/NADPH) treated anduntreated glutenins were incubated in 100 μl of simulated intestinalfluid (SIF) (Board of Trustees (ed.), 1995, Simulated Gastric Fluid,TS., pp 2053, The United States Pharmacopeia, 23, The National Formulary18, United States Pharmacopeial Convention, Inc., Rockville, Md.) asdescribed below. SIF contained 5 μg trypsin (or 20 μg pancreatin), 48.9mM monobasic potassium phosphate, and 38 mM sodium hydroxide. Afteraddition of the enzyme, the pH was brought to 7.5 with 0.2 M sodiumhydroxide. Digests were incubated in a 37° C. water bath for 0, 20, 60,or 80 minutes. To stop the reaction, 100 mM PMSF and leupeptin (1 μg/ml)was added for trypsin digests and 1 N HCl for pancreatin digests.SDS-PAGE analysis of the digested samples was performed in 8-16%gradient gels as described by Laemmli (1970). Gels of 1.5 mm thicknesswere developed for 16 hr at a constant current of 7 mA. SDS gels werestained with Coomassie brilliant blue R-250 in 10% acetic acid for 30min, and destained in 10% acetic acid for 30 min with the aid of amicrowave oven. Protein stained gels were captured by Gel Doc 1000. Thequantification of protein (optical density×mm×mm) on the gels wascarried out with a software program for image analysis-Multi-Analyst,version 1.0 (Bio-Rad). Relative digestion was expressed as thepercentage of zero time undigested protein.

The results shown in FIGS. 16 and 17 demonstrate that thioredoxinreduction results in enhanced susceptibility of glutenins to proteasedigestion by trypsin and pancreatin, respectively. The most pronouncedeffects were observed with trypsin where about 55% of protein remainedat 60 minutes post-digestion in the NTS treated sample in comparison toabout 90-95% of the starting protein remained in the non-NTS treatedsample. In the trypsin digestions, proteolysis progressed for 60 minutesand apparently plateaued. In the pancreatin digests, proteolysisprogressed less rapidly. At 80 minutes post-pancreatin treatment, about60% of the starting proteins remained in the NTS treated sample incomparison to 95% protein remaining in the non-NTS sample. Thus thetransgenic grains of the present invention are more susceptible todigestion and are hyperdigestible. The increase in the digestibility isat least 5% in the transgenic plants in comparison to the non-transgenicgrains.

Example 4 Effect of NTR on the Reduction of Proteins in Extracts ofWheat Grains Overexpressing Thioredoxin h

In Vitro Reduction of Proteins by NADPH or NTR or NADPH & NTR

Aliquots of the albumin/globulin fraction from the homozygous linesoverexpressing thioredoxin h as described in Example 2 and nullsegregant lines were used. The reaction was carried out in 30 mMTris-HCl buffer, pH 7.9. As indicated the treatments were: (i) control,(ii) 1.25 mM NADPH, (iii) 3.0 μg Arabidopsis NTR, (iv) NADPH & NTRcombined, and (v) 5 mM dithiothreitol (DTT). The above reagents wereadded to 70 microliters of this buffer containing 60 μg of protein.Total reduction by dithiothreitol (DTT) was achieved by boiling for 5min. After incubation for 60 min at 37° C., 100 nmoles of mBBr wereadded and the reaction was continued for another 15 min at roomtemperature. To stop the reaction and derivatize, excess mBBr, 10, μl of100 mM MET was added. The reduced samples, after adding 25 μl of 4×Laemmli sample buffer, were analyzed as described by mBBr/SDS-PAGE(Kobrehel, K. et al. 1992).

The results shown in FIG. 18 indicate that the albumin/globulin proteinsin the homozygous transgenics overexpressing thioredoxin h are moreefficiently reduced than the albumin/globulin fraction of grain fromtheir null segregant counterparts.

Example 5 Effect of Overexpressed Thioredoxin h on Allergenicity ofProteins From Wheat Grain

The following protocol was approved by the appropriate committees atboth the University of California-Davis (Animal Use and CareAdministrative Advisory Committee, effective Jan. 21, 1999-Jan. 21,2000) and the University of California-Berkeley (Animal Care and UseCommittee, effective May 1, 1999-Apr. 30, 2000).

Dogs from the UC-Davis sensitized Dog Colony (Ermel et al. 1997) thatwere sensitized to commercial whole wheat grain extract (Bayer), wereselected as strong reactors from two groups: 1) 2 year-old, designated“young dogs,” and 2) 7 year-old, “old dogs.” Before starting the skintests, each animal received an intravenous injection of 5 ml sterilesaline solution containing 0.5% Evans Blue (0.2 ml/kg). After 5 min,skin tests were performed by 100 μl intradermal injections of logdilutions of each wheat protein fraction in PBS buffer on the ventralabdominal skin. The quantity of protein injected ranged from 33 pg to 10μg. The fractions tested were: 1) salt water-soluble (albumins andglobulins); 2) ethanol-soluble (gliadins); acid acetic-soluble(glutenins). After 20 min, length and width of wheal areas were measuredby a blinded reader. The total area was calculated as an ellipse(π/4×L×W). Protein allergenicity of the null segregant (control) and thehomozygous wheat lines was obtained by comparison of the total whealarea generated by the different dilutions of each extract.

The responses of the animals are shown in FIG. 19 and indicate that theproteins obtained from the transgenic wheat are less allergenic that theprotein obtained from the null segregant. For each fraction tested, bothyoung and old animals were less responsive to proteins from transgenicwheat. The allergenicity with the transgenics were decreased at least 5%in comparison to nontransgenic controls. The allergencity in the youngdogs was more substantially reduced, ranging from 20 to 32% decrease. Incontrast, the allergenicity in older animals was reduced by 8 to 23%.

To demonstrate the hypoallergenicity of malt produced from thetransgenic wheat grain, malt is produced according to standard protocolsknown in the art from the transgenic seeds. Extracts of the malt areproduced according to the above procedure. Young and old sensitizeddogs, as described above, are injected intravenously with about 5 mlsterile saline solution containing 0.5% Evans Blue (0.2 ml/kg). Afterabout 5 min, skin tests are performed by 100 μl intradermal injectionsof log dilutions of each malt protein fraction in PBS buffer on theventral abdominal skin. The quantity of protein injected is about 33 μgto 10 μg. The fractions are as described above. After about 20 min, thelength and width of the wheal areas are measured by a blinded reader andthe total area is calculated as an ellipse. Malt protein allergenicityof malt produced from a null segregant (control) and malt fromhomozygous wheat lines are obtained by comparison of the total whealarea as described above. The allergenicity in the young dogs is moresubstantially reduced, and range from about 20-30% decrease. The olderanimals allergenicity is reduced by about 5-20%.

Accordingly, a food product such as beer produced from thehypoallergenic malt also is hypoallergenic.

Example 6 Transgenic Sorghum Expressing Barley Thioredoxin h

A. Seed Digestibility

Seeds from ten major cultivars of Sorghum vulgare are screened for athioredoxin-dependent increase in digestibility of constituent proteinsusing simulated gastric (pepsin), and intestinal (pancreatin) fluids.The cultivars are representative of those grown in the United States,Australia and different parts of Africa.

Albumin, globulin, kafirin. and glutelin protein fractions are isolatedaccording to their differential solubilities. Seed, 3 g, is ground in acoffee grinder, extracted sequentially with 30 ml of: [1] 0.5 M NaCl,[2] 60% (v/v) 2-propanol, and [3] 0.1 M sodium borate buffer, pH 10, ona shaker at 25° C. for 30 min, 4 hours, and 4 hours, respectively. Theextracted fractions correspond, respectively, to [1] albumin plusglobulin [2] kafirin, and [3] glutelin. Total kafirins or cross-linkedkafirins are extracted with 60% 2 propanol plus 1% 2-mercaptoethanol(Shull et al., 1992). Each suspension is clarified by centrifugation at10,000×g for 20 min at 4° C.; three successive extractions are performedwith the salt solution followed by two water washes. The remainingextractions are repeated twice. Resulting supernatant solutions arepooled and the digestibility of each fraction is tested on the same dayas isolation.

Aliquots of individual sorghum protein fractions are reduced either withthe NADP/thioredoxin or the NADP/glutathione system prior to digestionand the results compared with untreated control preparations.Alternatively, total protein extracted with sodium myristate, anonreducing detergent that solubilize wheat gliadins and glutenins in abiochemically active form (Kobrehel and Buchuk, 1978) can be tested fordigestibility. Reduction of the disulfide bonds of proteins is performedusing mBBr/SDS-PAGE as previously described (del Val et al., 1999) in avolume of 100 μl with either: (i) the NADP/thioredoxin system,consisting of 5 μl of 25 mM NADPH, 8 μl of 0.3 mg/ml E. coli thioredoxinand 7μl of 0.3 mg/ml E. coli NTR; or (ii) the NADP/glutathione systemcomposed 5 μl of 25 mM NADPH, 10 μl of 30 mM glutathione and 15 μl of0.1 mg/ml glutathione reductase. Reactions are carried out in a 30 mMphysiological buffered saline (PBS) solution containing 50 μg of eachprotein. The reaction mixtures are incubated at 4° C. overnight or at37° C. and 55° C. for 15 min (Kobrehel et al., 1992; del Val et al.,1999). The temperature found to work best is used for subsequentexperiments. For complete reduction, samples are incubated in PBS with 5μl 100 mM DTT and boiled 5 min. Protein fractions (albumin-globulin,kafirin, glutelin: 240 μg protein) is subjected to simulated digestion,either untreated or reduced with NADP/thioredoxin or NADP/glutathione,by pepsin (gastric simulation) or trypsin/chymotrypsin/carboxypeptidase(pancreatin: intestinal simulation).

Pepsin Assay

Each fraction, 500 μg of protein, is added to 100 μl of simulatedgastric fluid [0.32% pepsin (w/v) and 30 mM NaCl adjusted to pH 1.2 withHCl] (Astwood et al., 1996). The reaction mixture is incubated for up to60 min at 37° C. and stopped with 0.375-fold volume of 160 mM Na₂CO₃ togive neutral pH. The protein mixture is subjected to SDS-PAGE andstained for protein with Coomassie blue as described below.

Pancreatin Assay

Each fraction, 500 μg protein, is added to 100 μl of simulatedintestinal fluid (1% porcine pancreatin (w/v), 48.9 mM monobasicpotassium phosphate and 38 mM NaOH adjusted to pH 7.5 with NaOH) (seeUnited States Pharmacopeai, 1995). The reaction mixture is incubated forup to 60 min at 37° C. and stopped with {fraction (1/10)} volume of 100mM phenylmethyl sulfonyl fluoride (PMSF) plus 1 μg/ml leupeptin. Theprotein mixture is subjected to SDS-PAGE and stained with Coomassie blueas described below.

Two widely grown cultivar showing the most improved susceptibility toproteolytic and starch digestion after reduction by the thioredoxinsystem are used for the transformation work.

B. Isolation and Digestibility of Starch

Starch Granule Isolation

Starch granules from dry mature sorghum grain are extracted as described(Sun and Henson 1990). Sorghum grain is washed with distilled water andsteeped for 48 h in 20 mM Na-acetate buffer, pH 6.5, containing 0.02%NaAzide. Softened kernels are ground first with a motar and pestle andthen with a VirTis homogenizer for 6 min at 80% full speed and the gristpassed through two sieves (250 and 75 μm). Crude starch that passesthrough both sieves is purified by centrifugation (60×g for 2.5 min)through a layer of 65% (w/v) sucrose. Pelleted starch granules arerecentrifuged one or two times under the same conditions and resuspendedin 20 mM sodium acetate buffer, pH 6.5 containing 0.02% sodium azide.

Starch Digestion

Starch digestibility is measured based on enzymatic hydrolysis usingporcine pancreatic alpha-amylase (Type VI-B, Sigma Chemical Co., St.Louis, Mo.). Incubation mixtures containing 2% (w/v) starch, 0.5% (w/v)BSA, 0.02% (w/v) azide, 25 mM NaCl, 5 mM CaCl₂, and 10 units ofalpha-amylase in 10 mM sodium phosphate buffer, pH 6.9, are incubated37° C. Aliquots (50 to 100 μl) of reaction mixture is periodicallyremoved for determination of glucose and total reducing sugars releasedfrom starch granules. Reducing sugar concentration is measured by thedinitrosalicylic acid method (Bernfeld, 1955) and total starch contentby the enzymatic procedure of McClear et al., (1994).

Reduction of Protein on Starch Granules

Aliquots of the isolated 2% (w/v) starch are incubated with the NTSsystem to reduce the proteins on the surface of the granule as describedabove (Examples 3 and 4). Following reduction, the starch granules aretested for digestibility by alpha-amylase (McCleary et al., 1994) andstimulated intestinal fluid (Board of Trustees 1995)

C. Production of Stably Transformed Sorghum Lines and T₁ PlantsContaining Barley trxh

Using a cDNA library from scutellum tissues of barley (constructed by R.Schuurink, UCB), a full-length gene for thioredoxin h (trxh; FIG. 20)was isolated and characterized (Calliau, del Val, Cho, Lemeaux,Buchanan, unpublished). The full-length cDNA clone has been placed intoexpression vectors with the hordein promoters plus the targetingsequence as described (Cho et al., unpublished) is used for sorghumtransformation. This vector, pdBhssBTRXN-2, contains the 0.43-kbB₁-hordein promoter plus its signal sequence, barley trxh (btrxh) andnos.

Sorghum is transformed by the methods of Cho et al,(1998b, 1999b, 1999c,1999d, 2000) to give rise to highly regenerative green tissues. Thesetissues contain multiple, light-green, shoot meristem-like structures,which were characterized as such in barley because they expressed a geneassociated with maintenance of the shoot meristematic state, a knofted Ihomologue (Zhang et al., 1998). Target tissues such as these highlyregenerative tissues, which a high percentage of totipotent cellscapable of sustained cell division and competent for regeneration overlong period, represent a high-quality target tissue for transformation.They can be maintained for more than a year with minimal loss inregenerability (Cho et al., 1998b, 1999b, 1999c, 1999d, 2000; Kim etal., 1999; Ha et al., 2000). In addition, the result from genomic DNAmethylation analyses (Zhang et al. 1999b) showed that barley plantsregenerated from these highly regenerative tissues were less variable interms of methylation pattern polymorphism and agronomic performance thanthose regenerated from callus maintained in the embryogenic state.

Media developed for the other cereals and grasses are utilized foroptimizing the response of the sorghum variety, TX430, to produce highquality, green regenerative tissues with sorghum similar to thoseobserved with other cereals and grasses. Such tissues have been usedsuccessfully for stable transformation with all varieties tested.Briefly, this method, the development of green, regenerative tissues,involves the initiation of embryogenic cultures from immature embryos ofcultivar TX430. The medium giving the highest quality tissue is D′BC2and DBC3 (Cho et al., 1998a-c, 1999d). Such media, containing copper,maltose, and cytokinins have been found to improve the quality andlong-term regenerability of tissue from other cereal and grasses. Tissuedeveloped on this medium is used as transformation targets usingbombardment.

The desired DNA construct(s) containing barley trxh are introduced intotarget cells via bombardment. Selection to identify transformants is viabialaphos, kanamycin, or other appropriate selection agents according topublished procedures (Cho et al., 1998a-c; Lemaux et al. 1999). Smallportion of putatively transformed calli are analyzed by PCR (Cho et al.,1998a-c) for barley trxh and transformed tissue is manipulated toregenerate plants (Cho et al, 1998a-c). Leaf tissue is tested forresistance to the selective agent, if possible, and as appropriate isanalyzed by PCR for the transgene(s). Plants are grown to maturity toobtain T₁ seeds and homozygous T₂ plants.

D. Determination of Amounts and Activity of TRXh in Stably TransformedSorghum

The activity of the barley thioredoxin h from the different productionsystems (targeted vs. nontargeted, i.e, with or without the signalsequence, respectively) and obtained with different fractionationprocedures, as described above, is assayed using the DTNB[2′,5′-dithiobis (2-nitrobenzoic acid)] method (Florencio et al., 1988)as described (Cho et al., 1999e). The NTR and thioredoxin controls areprepared from wheat grains as described by Johnson et al., (1987a, b).

Western Blot Analysis

Western blots are performed on extracts from selected transgenic linesas well as control seeds. Lots of 10 to 20 intact seeds are processedand analyzed for content of TRXh and NTR by SDS-PAGE and western blotprocedures (Cho et al., 1999e).

Preparation of Seed Extract, Heat Treatment and Column Chromatography

Extracts are prepared, heat treated, and fractionated by columnchromatography as described by Cho et al., (1999e).

Measurement of Thioredoxin h Activity

Thioredoxin h is assayed by the chloroplast NADP-malate dehydrogenaseprocedure as adapted for barley (Cho et al., 1999).

Protein Determination

Protein is determined or measured according to Bradford (1976) using theCoomassie blue method with gamma-globulin as a standard. Protein contentis confirmed by measuring total nitrogen in an automated gas analyzer orby standard micro-kjeldahl procedure.

E. Measurements in Changes in Abundance and Redox State of EndospermProteins

Transgenic sorghum seeds overexpressing barley thioredoxin h are thestaring material used to demonstrate that increased levels of thisprotein cause altered digestibility. Preliminary mBBr measurements arealso made with the genetically engineered grain. Changes in the redoxstate of endosperm protein are determined using the mBBr/SDS-PAGEprocedure (Krobehel et al., 1992). As the major indigenous storageproteins in sorghum are known to be insoluble, propanol as well as thedifferent aqueous endosperm extracts are monitored in the grain.Residues are extracted sequentially, as described above (A. SeedDigestibility) for the various protein fractions. Supernatant fractionsof each extract is analyzed for protein and fluorescence by themBBr/SDS-PAGE technique.

Dry grain, 1 g, from transgenic and null segregant lines are ground witha mortar and pestle in liquid nitrogen. When the liquid nitrogenevaporates, 3-6 ml of 30 mM Tris-HCl, pH 7.9 buffer containing 1 mM EDTAand 1 mM mBBr is added and mixed for 1 min. After thawing the extract isincubated 15 min, centrifuged (10 min at 12,000×g), extractedsequentially with salt, propanol, and borate solutions as describedabove (A. Seed Digestibility). Sixty μg protein samples are loaded ontoa 10-20% SDS-polyacrylamide gradient gel as described above. Followingelectrophoresis (1 h, constant current of 30 mA), gels are soaked for 2h in 12% (w/v) trichloroacetic acid and transferred to a solutioncontaining 40% methanol and 10% acetic acid for 12 h to remove excessmBBr. Gels are scanned for fluorescence with a UV light source (365 nm)and stained for protein with Coomassie blue.

F. Measurements of Change in Digestibility and Solubility of EndospermProteins in T₁ Heterozygous and T₂ Homozygous Sorghum Grain

In parallel with the in vitro experiments (Ori et al., 1995), the extentthat in vivo thioredoxin-mediated reduction contributes to thedigestibility and solubility of sorghum endosperm proteins isdetermined. The extent of solubilization of protein is measured usingthe ratio of the soluble to the insoluble protein in the transgenic,relative to a null segregant. Extracts are prepared in parallel withoutmBBr labeling and tested for susceptibility to digestion by simulatedgastric and intestinal fluids are described above (Example 3). Theproteins from the different transgenic grain also are reduced withthioredoxin and glutathione as described above (A. Seed Digestibility).

G. Measurements of Change in Digestibility of Starch in T₁ Heterozygousand T₂ Homozygous Sorghum Grain

As in the case of the kafirin storage proteins, the ability of theoverexpressed thioredoxin h to enhance the digestibility of starch withalpha-amylase is determined. The starch is isolated from both transgenicand null segregant lines and its digestibility tested in vitro withalpha-amylase as described above (B. Isolation and Digestibility ofStarch). Because of their association with starch granules, an increasein the digestibility of the kafirin proteins is accompanied by anincrease in the digestibility of the starch.

H. Thioredoxin h Overexpressed in Sorghum to Improve Digestibility ofGrain Protein

The above-noted digestibility of the different protein fractions(albumin/globulin, kafirin, glutelin) is tested with simulated gastricand intestinal fluids. The results from the transgenic grainoverexpressing barley TRXh is compared to those with the null segregantto demonstrate improvement in digestibility in the transgenic grain.

Example 7 Improvement of Dough Quality

In U.S. application Ser. No. 08/211,673 (expressly incorporated byreference), dough quality was improved by reducing the flour proteinsusing the NADP/thioredoxin system. Without being bound by theory,reduced thioredoxin specifically breaks intramolecular sulfur-sulfurbonds that cross-link different parts of a protein and stabilize itsshape. When these cross-links are broken the protein can unfold andsupposedly link with other proteins in dough, creating an interlockinglattice that forms an elastic network. The dough rises because thenetwork helps trap carbon dioxide produced by yeast during thefermentation process. It was proposed that the reduced thioredoxinreduced the gliadins and glutenins in flour letting them recombine in away that strengthened the dough. Reduced thioredoxin facilitated theirforming a protein network during dough making. Treatment of intermediateor poor quality wheat flour (Apollo cultivar) with E. coli thioredoxin,NADP-thioredoxin reductase, and NADPH showed dough strengthening (higherfarinograph measurements) and improved loaf volume and viscoelasticityin comparison with untreated flour. Higher farinograph measurements ofdough correspond to improved dough strength and improved baked goodcharacteristics such as better crumb quality, improved texture andhigher loaf volume.

Wheat Bread Baking Studies and Farinograph Measurements

The baking tests are carried out by using a computer operated PANASONICbread maker to demonstrate improved quality of dough made using flourprepared from the transgenic seeds of the present invention.

Composition of bread:

Control: Flour*: 200 gm (dry) Water: 70% hydratation Salt (NaCl): 5.3 gYeast: 4.8 g (S. cerevisiae) (dry yeast powder)

*Flour samples are obtained from transgenic and non-transgenic wheat(cv. Thesee, Apollo, Arbon, and other animal feed grade and other gradeshaving from poor to good baking quality), sorghum, corn, and rice.

Experimental Conditions

-   -   Flour and salt are weighed and mixed    -   The volume of water needed to reach a hydration of 70% was put        into the bread maker.    -   The mixture of flour and salt is added to the water and the        baking program is started by the computer. The complete program        lasts about 3 hrs 9 min and 7 secs.    -   Yeast is added automatically after mixing for 20 min and 3 secs.

The program operating the Panasonic apparatus is: Mixing SegmentsDuration Conditions Heating Mixing 00:00:03 T1 off Mixing 00:05:00 T2off Mixing 00:05:00 T1 off Rest 00:10:00 TO off Mixing 00:17:00 T2 offMixing 00:07:00 T1 off Rest 00:30:00 TO to reach 32° C. Mixing 00:00:04T1  32° C. Rest 01:15:00 TO  32° C. Baking 00:14:00 TO to reach 180° C.Baking 00:26:00 TO 180° C.Mixing Conditions:TO = no mixing (motor at rest)T1 = normal mixingT2 = alternately 3 second mixing, 3 second restAfter the dough is formed, farinograph readings are taken as describedin U.S. application Ser. No. 08/211,673. Bread loaf volume is measuredat the end of the baking, when bread loaves reach room temperature.Farinograph readings of dough produced from flour made from transgenicwheat seeds of the invention are at least about 10-20% higher and aremaintained about 40% longer than dough produced from flour made fromnon-transgenic seeds. Bread produced from flour made from transgenicseeds of the invention has at least about 5% and up to about 20%increased volume in comparison to bread produced from flour made fromnon-transgenic seeds. Bread-like products made from transgenic flour ofcereals that normally produce a nonglutenous flour, for example, rice,hold together and hold gas better than products produced from the flourof their nontransgenic counterparts. They also show at least a 3%increase in loaf volume when compared to their nontransgeniccounterparts.

Example 8 Effect of Glucose-6-Phosphate Dehydrogenase on Reduction ofProteins in Exacts of Homozygous vs. Null Segregant Wheat GrainOverexpressing Thioredoxin h

Samples were from the salt-soluble fractions (albumin and globulin) ofthe transgenic and null segregant wheat grain overexpressing wheatthioredoxin h. Reactions were carried out in 30 mM Tris-HCl buffer, pH7.9, in a final volume of 100 μl. The complete reaction mixturecontained 10 μmol glucose-6-phosphate, 0.25 μmol NADP, 2 unitsglucose-6-phoshate dehydrogenase (Bakers Yeast, Type XV, Sigma, St.Louis, Mo.), plus or minus 1.5 μg NTR (Arabidopsis), and 80 μg protein.Other treatments, where omission of one or two component(s) of the NADPHgenerating system, were as indicated. The negative control was theextracted protein alone. As a positive control NADPH was used in placeof NADP/glucose-6-phoshate/glucose-6-phosphate dehydrogenase.

After incubation at 37° C. for 60 min, 100 nmol mBBr was added tot hereaction mixture, and the reaction was continued for 15 min. Ten μl of100 mM 2-mercaptoethanol was added to stop the reaction and derivatizeexcess mBBr. An appropriate amount of 4× Laemmeli sample buffer wasadded and the samples were applied onto 10-20% polyacrylamide gel in thepresence of SDS. Electrophoresis was carried out at room temperature at7 mA/gel for 16 hours. Flourescence of sulfhydryl containing proteins ongels was captured by Gel Doc 1000 (Blo-Rad), protein was stained by0.025% Coomassie Brilliant Blue G-250 in 10% acetic acid.

For visualizing the effect of glucose-6-phosphate dehydrogenase (FIG.23): in the presence of NTR, comparison of lanes 2 vs. 4 (−NADP) andlanes 5 vs 7 (+NADP) (+NTR gel on the left); in the absence of NTR,compare lanes 1 vs. 3 (−NADP) and lanes 2 vs. 4 (+NADP) (−NTR gel on theright). The maximal increase in reduction effected byglucose-6-phosphate dehydrogenase was observed in the presence of NTR,without NADP (lane 2 vs. lane 4, gel on the left). Note also the greaterreduction of NTR in lane 4 vs. lane 2.

With the null segregant (FIG. 24), note the greater reduction of NTR inthe presence of glucose-6-phosphate dehydrogenase (lane 4 vs. lane 2)but a lower extent of the reduction of the smaller target proteins (lane4) compared to the corresponding treatment (lane 4) with the transgenicextract (FIG. 23).

This invention has been detailed both by example and by description. Itshould be apparent that one having ordinary skill in the relevant artwould be able to surmise equivalents to the invention as described inthe claims which follow but which would be within the spirit of theforegoing description and examples. It should be realized that thoseequivalents and various modifications as may be apparent to those ofskill in the art to which the invention pertains also fall within thescope of the invention as defined by the appended claims. All hereincited patents, patent applications, publications, references, andreferences cited therein are hereby expressly incorporated by referencein their entirety.

References

All references, patents, patent applications, publications, andreferences cited therein are hereby expressly incorporated by referencein their entirety.

-   -   Altschul et al. (1990) J. Mol. Biol. 215:403-10.    -   Altschul et al. (1994) Nature Genet. 6:119-29.    -   Altschul et al. (1996) Methods in Enzymology 266:460-480.    -   Astwood et al. (1996) Nature Biotech. 14:1269-1273.    -   Ausubel et al. (1987) In: Current Protocols in Molecular        Biology, Greene Publishing Associates and Wiley-Intersciences.    -   Bagga et al. (1997) Plant Cell 9:1683-1696.    -   Bernfeld P (1955) Amylases, alpha and beta. Methods Enzymol.        1:149-158    -   Besse and Buchanan (1997) Bot. Bull. Acad. Sin. (Taipei)        38:1-11.    -   Besse I, Wong J H, Kobrehel K, Buchanan B B (1996) Thiocalsin: A        thioredoxin-linked, substrate-specific protease dependent on        calcium. Proc. Natl. Acad. Sci. USA 93:3169-3175.    -   Berstermann A, Vogt K, Follmann H (1983) Plant seeds contain        several thioredoxins of regular size. Eur/J/ Biochem.        131:339-344.    -   Bondenstein-Lang J, Buch A, Follman H (1989) Animal and plant        mitochondria contain specific thioredoxins. FEBS Lett.        258:22-26.    -   Bower M S, Matias D D, Fernandes-Carvalho E, Mazzurco M, Gu T,        Rothstein S J, Goring D R (1996) Two members of the thioredoxin        in-family interact with the kinase domain of a Brassica S locus        receptor kinase. Plant Cell 8:1641-1650.    -   Bradford M M (1976) Anal. Biochem. 72:2 48-254.    -   Brandt A, Montembault A, Cameron-Mills V, Rasmussen S K (1985)        Primary structure of a B₁ hordein gene from barley. Carlsberg        Res. Commun. 50:333-345.    -   Brugidou C, Marty I, Chartier Y, Meyer Y (1993) The Nicotiana        tabacum genome encodes two cytoplasmic thioredoxin genes which        are differently expressed. Mol. Gen. Genet. 238:285-293.    -   Buchanan B B (1991) Regulation of CO₂ assimilation in oxygenic        photosynthesis: the ferredoxin/thioredoxin system. Arch.        Biochem. Biophys. 287:337-340.    -   Buchanan B B, Adamidi C, Lozano R M, Yee B C, Momma M, Kobrehel        K, Ermel R Frick O L (1997) Thioredoxin-linked mitigation of        allergic responses to wheat. Proc. Natl. Acad. Sci. USA        94:5372-5377.    -   Buchanan et al. (1994) Arch. Biochem. Biophys. 314:257-260.    -   Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996)        Engineered GFP as a vital reporter in plants. Current Biol.        6:325-330.    -   Cho M-J, Lemaux P G (1997a) Rapid PCR amplification of chimeric        products and its direct application to in vivo testing of        recombinant DNA construction strategies. Mol. Biotechnol.        8:13-16.    -   Cho M-J, Vodkin I, Widholm J M (1997b) Transformation of soybean        embryogenic culture by microprojectile bombardment. Plant        Biotechnol. 14:11-16.    -   Cho M-J, Ha C D, Buchanan B B, Lemaux P G (1998a) Subcellular        targeting of barley hordein promoter-uidA fusions in transgenic        barley seed. P-1024. Congress In Vitro Biology, Las Vegas, Nev.        30 May-3 Jun. 1998.    -   Cho M-J, Jiang W, Lemaux P G (1998b) Transformation of        recalcitrant cultivars through improvement of regenerability and        decreased albinism. Plant Sci. 138:229-244.    -   Cho M-J, Zhang S, Lemaux P G (1998c). Transformation of shoot        meristem tissues of oat using three different selectable        markers. In Vitro Cell Dev. Biol. 34P:340    -   Cho M-J, Choi H W, Buchanan B B, Lemaux P G (1999a) Inheritance        of tissue-specific expression of barley hordein promoter-uidA        fusions in transgenic barley plants. Theor. Appl. Genet.        98:1253-1262.    -   Cho M-J, Buchanan B B, Lemaux P G (1999b) Development of        transgenic systems for monocotyledonous crop species and        production of foreign proteins in transgenic barley and wheat        seeds. In: Application of Transformation Technology in Plant        Breeding. Special Seminar for the 30th Anniversary Korean        Breeding Soc., Suwon, Korea, Nov. 19, 1999, pp. 39-53.    -   Cho M-J, Choi H W, Lemaux P G (1999c) Transgenic orchardgrass        (Dactylis glomerata L.) plants produced from high regenerative        tissues. P-1089. Congress In Vitro Biology, New Orleans, La. 5-9        Jun. 1999.    -   Cho M-J, Jiang W, Lemaux P G (1999d) High frequency        transformation of oat via microprojectile bombardment of        seed-derived regenerative cultures. Plant Sci. 148:9-17.    -   Cho M-J, Wong J, Marx C, Jiang W, Lemaux P G, Buchanan B B        (1999e) Overexpression of thioredoxin h leads to enhanced        activity of starch debranching enzyme (pullulanase) in        germinating barley seeds. Proc. Natl. Acad. Sci. USA        96:14641-14646.    -   Cho M-J, Ha C D, Lemaux P G (2000) Production of transgenic tall        fescue and red fescue plants by particle bombardment of mature        seed-derived highly regenerative tissues. Plant Cell Rep. (in        press).    -   Christensen and Quail (1996) Transgenic Res. 5:1-6.    -   Conrad etal. (1998) Journal of Plant Physiology 152:708-711.    -   Corpet et al. (1988) Nucleic Acids Research 16:10881-90. del        Val, Yee B C, Lozano R M, Buchanan B B, Ermel R E, Lee Y M, and        Frick O L (1999) J. Aller. Clin. Immunol. 103:690-697.    -   Dai S, Saarinen M, Ramaswamy S, Meyer Y, Jacquot J-P, Eklund        H (1996) Crystal structure of Arabidopsis thaliana NADPH        dependent thioredoxin reductase at 2.5 Å resolution. J. Mol.        Biol. 264:1044-1057.    -   Dellaporta S (1993) Plant DNA miniprep and microprep. Freeling        M, Walbot V (eds) In: Maize Handbook. p 522-525.    -   Del Val G, Yee B C, Lozano R M, Buchanan B B, Ermel R W, Lee Y        M, Frick O L (1999). Thioredoxin treatment increases        digestibility and lower allergenicity of milk. J. Allergy        Clinical Immunol. 103(4):690-697.    -   Entwistle J, Knudsen S, Muller M, Cameron-Mills V (1991) Amber        codon suppression: the in vivo and in vitro analysis of two        C-hordein genes from barley. Plant Mol. Biol. 17:1217-1231.    -   Ermel R W, Knock M, Griffe S M, Reinhart G A, Frick O L. 1997.        The atopic dog; A mode for food allergy. Lab. Animal Sci.        47:40-49.    -   Fennema O R (1996) Food Chemistry. New Marcel Dekker. 1-15    -   Florencio et al. (1988) Arch. Biochem. Biophys. 266:496-507.    -   Forde B G, Heyworth A, Pywell J, Kreis M (1985) Nucleotide        sequence of a B1 hordein gene and the identification of possible        upstream regulatory elements in endosperrn storage protein genes        from barley, wheat and maize. Nucl. Acids Res. 13:7327-7339.    -   Furlong-Munoz A (1996) The basis of food allergies. Cereal Food        World. 41:71-72.    -   Gautier et al. (1998) Eur. J. Biochem. 252:314-324.    -   Gelvin et al., (1990) Plant Molecular Biology Manual, Klower        Academic Publishers.    -   Grimwade et al. (1996) Plant Molecular Biology 30:1067-1073.    -   Ha C D, Lemeaux P G, Cho M-J (2000) Stable transformation of a        recalcitrant Kentucky bluegrass (Poa praetensis L.) cultivar        using mature seed derived high regenerative tissues. In Vitro        Cell Dev. Biol. (submitted)    -   Hamaker B R, Kirleis A W, Mertz E T, Axtell J D (1986) Effect of        cooking on the protein profiles and in vitro digestibility of        sorghum and maize. J. Agric. Food Chem. 34:647-649.    -   Hardie D G. (1975) Phytochem. 14:1719-1722.    -   Higgins and Sharp (1988) Gene 73:237-244.    -   Higgins and Sharp (1989) CABIOS 5:151-153.    -   Huang X et al. (1992) Parallelization of a local similarity        algorith. Compt. Appl. Biosci. 8:155-165.    -   Hunter C P (1988) Plant regeneration from microspores of barley,        Hordeum vulgare. PhD thesis. Wye College, University of London,        Ashford, Ky.    -   Innis et al. (eds.) (1990) PCR Protocols, A Guide to Methods and        Applications, Academic Press, Inc., San Diego, Calif.    -   Ishiwatari et al. (1995) Planta 195(3):456463.    -   Jacquot J-P, Rivera-Madrid R, Marino P, Kollarova M, Le Maréchal        P, Miginiac-Maslow M, Meyer Y (1994) Arabidopsis thaliana        NADPH-thioredoxin reductase cDNA characterization and expression        of the recombinant protein in Escherichia coli. J. Mol. Biol.        235:1357-1363.    -   Jiao J, Yee B C, Kobrehel K, Buchanan B B (1992) Effect of        thioredoxin-linked reduction on the activity and stability of        the Kunitz and Bowman-Birk soybean trypsin inhibitor        proteins. J. Agric. Food Chem 40:2333-2336.    -   Johnson T C, Cao R Q, Kung J E, Buchanan B B, Holmgren (1987a)        Thioredoxin and NADP-thioredoxin reductase from cultured carrot        cells. Planta 171:321-331.    -   Johnson T C, Wada K, Buchanan B B, Holmgren A (1987b) Reduction        of purothionin by the wheat seed thioredoxin system and        potential function as a secondary thiol messenger in redox        control. Plant Physiol. 85:446-451.    -   Kim H-K, Lemaux P G, Buchanan B B, Cho M-J (1999) Reduction of        genotype limitation in wheat (Triticum aestivum L.)        transformation. P-1021. Congress In Vitro Biology, New Orleans,        La. 5-9 Jun. 1999.    -   Kobrehel and Bushuk (1978) Studies of Glutenin XI. Note on        Glutenin Solubilization and Surfactants in Water. Cereal        Chemistry 55:1060.    -   Kobrehel et al. (1994) Thioredoxin-linked reduction of wheat        storage proteins. II. Technological Consequences. In: Gluten        Proteins:1993. Association of Cereal Research; Detmold, Germany.    -   Kobrehel K, Wong J H, Balogh A, Kiss F, Yee B C, Buchanan B        B (1992) Specific reduction of wheat storage proteins by        thioredoxin h. Plant Physiol. 99:919-924.    -   Kobrehel K, Yee B C, Buchanan B B (1991) Role of the        NADP/thioredoxin system in the reduction of ct-amylase and        trypsin inhibitor proteins. J. Biol. Chem. 266:16135-16140.    -   Kruger et al., (1988) Cereal Chem. 65:208-214.    -   Kuriyan J. Krishna T S R, Wong L, Guenther B, Pahler A, Williams        C H, Model P (1991) Convergent evolution of similar function in        2 structurally divergent enzymes. Nature 352:172-174.    -   Laemmli U K (1970) Cleavage of structural proteins during the        assembly of the head of bacteriophage T4. Nature 227:680-685.    -   Lemaux P G, Cho M-J. Zhang S, Bregitzer P (1999) Transgenic        cereals: Hordeum vulgare (barley). Vasil I K (ed) In: Molecular        Improvement of Cereal Crops. Kluwer Academic Pub, pp 263-267    -   Lasztity R. 1996. The Chemistry of Cereal Proteins (2nd        edition). I. New York: CRC Press.    -   Lehrer S B, Horner W E, Reese G. (1996) Why are some proteins        allergenic? Implications for biotechnology. Crit. Rev. Food Sci.        Ntr. 36-553-564.    -   Lemaux P G, Cho M-J, Louwerse J, Williams R, Wan Y (1996)        Bombardment-mediated transformation methods for barley. Bio-Rad        US/KG Bulletin 2007:1-6.    -   Lemaux P G, Cho M-J, Zhang S, and Bregitzer P (1999) Transgenic        Cereals: Hordeum vulgare L. (barley). In: Molecular Improvement        of Cereal Crops. Ed. I.K. Vasil, 255-316, Kluwer Academic Publ.        UK.    -   Li X, Nield J, Hayman D, Langridge P (1995) Thioredoxin activity        in the C terminus of Phalaris S protein. Plant J 8:133-138.    -   Liu S, Kriz, A (1996) Tissue-specific and ABA-regulated maize        Glb1 gene expression in transgenic tobacco. Plant Cell Pep.        16:158-162.    -   Lozano R M, Wong J H, Yee B C, Peters A, Kobrehel K, Buchanan B        B (1996) New evidence for a role for thioredoxin h in        germination and seedling development. Planta 200:100-106.    -   Lozano R M, Yee B C, Buchanan B B (1994) Thioredoxin-linked        reductive inactivation of venom neurotoxins. Arch Biochem        Biophys 309:356-362.    -   MacGregor A W, Macri J L, Schroeder S W, Bazin S L (1994a) Limit        dextrinase from malted barley: Extraction, purification, and        characterization. Cereal Chem. 71:610-617.    -   MacGregor A W, Macri L J, Schroeder S W, Bazin S L (1994b) J.        Cereal Sci. 20:3341.    -   MacLean W C, Lopez-deRomano, Placho R P, Gaham G G (1981)        Protein quality and digestibility of sorghum in preschool        children. Balance Studies and Plasma Free Amino Acids. J. Nutr        111:1928-36.    -   Macri J L, MacGregor, A W, Schroeder, S W, Bazin, S L. (1993) J.        Cereal. Sci. 18:103-106.    -   Marcus F, Chamberlain S H, Chu C, Masiarz F R, Shin S, Yee B C,        Buchanan, B B (1991) Plant thioredoxin h: an animal-like        thioredoxin occurring in multiple cell compartments. Arch.        Biochem. Biophys. 287:195-198.    -   Mark and Richardson (1976) Proc. Natl. Acad. Sci. USA        73:780-784.    -   Marris C, Gallois P, Copley J, Kreis M (1988) The 5′ flanking        region of a barley B hordein gene controls tissue and        developmental specific CAT expression in tobacco plants. Plant        Mol. Biol. 10:359-366.    -   Marty I, and Meyer Y (1991) Nucleotide sequence of a        complementary DNA encoding a tobacco thioredoxin. Plant Mol.        Biol. 17:143-148.    -   Marx C, Lemaux P G, Buchanan B B (2000) The wheat grain: new        research developments and approaches to improvement. In: Seed        Technology. Eds. M. Black and D. Bewley, Sheffield Academic        Press, UK (in press)    -   McCleary et al. (1994) Quantitative measurement of total starch        in cereal products. J. Cereal Science 20:51-58.    -   Mena M, Vicente-Carbajosa J, Schmidt R J and Carbonero P (1998)        An endosperm-specific DOF protein from barley, highly conserved        in wheat, binds to and activates transcription from the        prolamin-box of a native B-hordein promoter in barley endosperm.        Plant Journal 16:53-62.    -   Mertz E T, Hassen M M, Cairns-Whittern C, Kirleis A W, Tu I,        Axtell J D (1984) Pepsin digestibility of proteins in sorghum        and other major cereals. Proc. Nati. Acad. Sci USA 81:1-2.    -   Muller M, Knudsen S (1993) The nitrogen response of a barley        C-hordein promoter is controlled by positive and negative        regulation of the GCN4 and endosperrn box. Plant J. 4:343-355.    -   Needleman and Wunsch (1970) J. Mol. Biol. 48:443.    -   Ori M P, Hamaker B R, Schull J M (1995) In vitro protein        digestibility of developing and mature sorghum grain in relation        to alpha-, beta-, and gamma-kafirin disulfide crosslinking.        Cereal Science 22:85-93.    -   Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:9444.    -   Pearson et al. (1994) Methods in Molecular Biology 24:307-331.    -   Rasmussen S K, Brandt A (1986) Nucleotide sequences of cDNA        clones for C-hordein polypeptides. Carlsberg Res. Commun.        51:371-379.    -   Rivera-Madrid et al. (1993) Plant Physiology 102:324-328.    -   Rivera-Madrid et al. (1995) Proc. Natl. Acad. Sci. USA        92:5620-5624.    -   Russel M, Model P (1988) Sequence of thioredoxin reductase from        Escherichia coli. Relation to other flavoprotein disulfide        oxidoreductases. J. Biol. Chem. 263:9015-9019.    -   Sambrook et al. (1989) In: Molecular Cloning. A Laboratory        Manual, Cold Spring Harbor, N.Y.    -   Scheibe R (1991) Redox-modulation of chloroplast enzymes. A        common principle for individual control. Plant Physiol. 96:1-3.    -   Schull J M, Watterson J J, Kirleis A W (1992) Purification and        immunocytochemical localization of kafirins in Sorghum bicolor        (L. Moench) endosperm. Protoplasma 171:64-74.    -   Serre L, Lauriere C. (1990) Analytical Biochemistry.        186(2):312-315.    -   Shewry P R, Field J M, Kirkman M A, Faulks A J, Miflin        B J. (1980) J. Exp. Botany 31:393-407.    -   Shewry et al. (1986) J. Cereal Sci. 4:97.    -   Shi J and Bhattacharyya M K (1996) A novel plasma membrane-bound        thioredoxin from soybean. Plant Mol. Biol. 32:653-662.    -   Sicherer S H (1999) Manifestation of food allergy: Evaluation        and management. Ameri. Fam. Physician. 59:415-424; 429-430.    -   Sissons M J, Lance R C M, Sparrow DHB. (1993) J. Cereal Sci.        7:19-24.    -   Sissons M J, Lance R C M, Wallace W. (1994) Cereal Chemistry.        71:520-521.    -   Smith and Waterman (1981) Adv. Appl. Math. 2:480.    -   Sørensen M B, Muller M, Skerritt J, Simpson D (1996) Hordein        promoter methylation and transcriptional activity in wild-type        and mutant barley endosperm. Mol Gen Genet 250:750-760.    -   Stemmer et al. (1994a) Nature 370:389-391.    -   Stemmer et al. (1994b) Proc. Natl. Acad. Sci. USA        91(22):10747-10751.    -   Sun Z, Henson C A (1990) Degradation of native starch granules        by barley alpha-glucosidases. Plant Physiology 94:320-327.    -   Suske G, Wagner W, Foliman H (1979) NADPH thioredoxin reductase        and a new thioredoxin from wheat. Z Naturforsch. C 34:214-221.    -   Takaiwa et al. (1995) Plant Science 111:39-49.    -   Torrent et al. (1997) Plant Molecular Biology 34:139-149.    -   Vollbrecht E, Veit B, Sinha N, Hake S. (1991) The development        gene Knotted-1 is a member of a maize homeobox gene family.        Nature 350:241-243.    -   Vogt K, Follmann H (1986) Characterization of three different        thioredoxins in wheat. Biochem Biophys Acta 873:415-418.    -   Wan and Lemaux (1994) Plant Physiol. 104:37-48.    -   Weissbach & Weissbach (1989) Methods for Plant Molecular        Biology, Academic Press.    -   Wong J H, Jiao J A, Kobrehel K, Buchanan B. (1995) Plant        Physiol. 108:67.    -   Wu et al. (1998) Plant Journal 14:673-683.    -   Zhang G, Hamaker D (1998) Low alpha-amylase starch digestibility        of cooked sorghum flours and the effect of protein. Cereal        Chemistry 75:710-713.    -   Zhang S, Williams-Carrier T, Jackson D, Lemaux P G. 1998.        Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot        meristem proliferation and adventitious shoot meristem formation        in maize (Zea mays L.) and barley (Hordeum vulgare L.). Planta        204:542-549.    -   Zhang S, Cho M-J, Koprek T, Yun R, Bregtizer P, and Lemaux P G.        (1999a) Genetic transformation of commercial cultivars of oat        (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro        shoot meristematic cultures derived from germinated seedlings.        Plant Cell Reports 18:959-956.    -   Zhang S, Zhang S, Cho M-J, Bregitzer P, and Lemaux P G. (1999a)        Comparative analysis of genomic DNA methylation status and field        performance of plants derived from embryogenic calli and shoot        meristematic cultures. In: Plant biotechnology and In Vitro        Biology in the 21st Century. Ed. A. Altman, 263-267. Kluwer        Academic Publishers, the Netherlands.    -   Zheng et al. (1995) Plant Physiology 109:777-786.

1-31. (Canceled).
 32. A transgenic monocot plant wherein at least partof said plant comprises a recombinant nucleic acid comprising a promoteractive in said part operably linked to a nucleic acid molecule encodinga thioredoxin h polypeptide wherein said promoter is a seed or grainmaturation-specific promoter.
 33. The transgenic plant of claim 32wherein said part is a seed.
 34. The transgenic plant of claim 32wherein said part is a grain.
 35. (Canceled).
 36. The transgenic plantof claim 32 wherein said promoter is selected from the group consistingof rice glutelins, rice oryzins, rice prolamines, barley hordeins, wheatgliadins, wheat glutelins, maize zeins, maize glutelins, oat glutelins,sorghum kasirins, millet pennisetins, rye secalins, and a maizeembryo-specific globulin promoter.
 37. The transgenic plant of claim 36wherein said barley hordein promoter is selected from the groupconsisting of 131 hordein and D hordein promoters.
 38. The transgenicplant of claim 32 wherein said plant is selected from the groupconsisting of rice, barley, maize, wheat, oat, rye, sorghum, millet,triticale, turf grass and forage grass.
 39. (Canceled).
 40. Thetransgenic plant of claim 32 wherein said thioredoxin h is barley,wheat, tobacco, rice, Brassica, Arabidopsis, Picea, or soy beanthioredoxin h.
 41. The transgenic plant of claim 32 wherein saidrecombinant nucleic acid further comprises a nucleic acid moleculeencoding a signal peptide operably linked to said promoter and saidnucleic acid molecule encoding a thioredoxin protein.
 42. The transgenicplant of claim 41 wherein said signal peptide targets expression of thethioredoxin polypeptide to an intracellular body.
 43. The transgenicplant of claim 42 wherein said signal peptide is selected from the groupconsisting of barley B1 hordein and D hordein signal peptides. 44-76.(Canceled).
 77. A transgenic seed or grain comprising a recombinantnucleic acid comprising a promoter active in said seed or grain operablylinked to a nucleic acid molecule encoding thioredoxin h polypeptidewherein said promoter is a seed or grain maturation-specific promoter.78. (Canceled).
 79. The transgenic seed or grain of claim 77 whereinsaid promoter is selected from the group 74 consisting of riceglutelins, rice oryzins, rice prolamines, barley hordeins, wheatgliadins, wheat glutelins, maize zeins, maize glutelins, oat glutelins,sorghum kasirins, millet pennisetins, rye secalins, and a maizeembryo-specific globulin.
 80. The transgenic seed or grain of claim 79wherein said barley hordein promoter is selected from the groupconsisting of B1 hordein and D hordein promoters.
 81. The transgenicseed or grain of claim 80 wherein said seed or grain is selected fromthe group consisting of rice, barley, maize, wheat, oat, rye, sorghum,millet, and triticale seed or grain.
 82. (Canceled).
 83. The transgenicseed or grain of claim 77 wherein said thioredoxin h is barley, wheat,tobacco, rice, soy bean, Brassica, Picea, or Arabidopsis thioredoxin h.84. The transgenic seed or grain of claim 77 wherein said recombinantnucleic acid further comprises a nucleic acid molecule encoding a signalpeptide operably linked to said promoter and said nucleic acid moleculeencoding a thioredoxin protein.
 85. The transgenic seed or grain ofclaim 84 wherein said signal peptide targets expression of thethioredoxin polypeptide to an intracellular body.
 86. The transgenicseed or grain of claim 85 wherein said signal peptide is selected fromthe group consisting of barley B1 hordein and D hordein signal peptides.87-111. (Canceled).
 112. The transgenic plant of claim 32 wherein saidplant is wheat.
 113. The transgenic plant of claim 112 wherein saidthioredoxin is wheat thioredoxin h.
 114. The transgenic seed or grain ofclaim 81 wherein said seed or grain is barley.
 115. The transgenic seedor grain of claim 114 wherein said thioredoxin is barley thioredoxin h.116. The transgenic seed or grain of claim 81 wherein said seed or grainis wheat.
 117. The transgenic seed or grain of claim 116 wherein saidthioredoxin is wheat thioredoxin h.
 118. The transgenic plant of claim32 wherein said thioredoxin is Arabidopsisthioredoxin h.
 119. Thetransgenic plant of claim 32 wherein said thioredoxin is soybeanthioredoxin h.
 120. The transgenic monocot seed or grain of claim 77wherein said thioredoxin is Arabidopsis thioredoxin h.
 121. Thetransgenic monocot seed or grain of claim 77 wherein said thioredoxin issoybean thioredoxin h.
 122. The transgenic plant of claim 32 whereinsaid thioredoxin is tobacco thioredoxin h.
 123. The transgenic plant ofclaim 32 wherein said thioredoxin is Brassica thioredoxin h.
 124. Thetransgenic monocot seed or grain of claim 77 wherein said thioredoxin istobacco thioredoxin h.
 125. The transgenic monocot seed or grain ofclaim 77 wherein said thioredoxin is brassica thioredoxin h.